Antiviral supramolecules containing target-binding molecules and therapeutic molecules bound to spectrin

ABSTRACT

Complexes are prepared containing two or more different effector molecules joined to each other by a joining component. One effector molecule is a binding molecule such as an antibody or Fc receptor that binds to a molecular target such as a virus or antibody at a site of infection or tumor, and another effector molecule is a therapeutic molecule such as an enzyme or drug. The joining component may be a liposome, protein or an organic polymer (including a dendrimer type polymer), and may be of sufficient length and/or flexibility to permit the therapeutic molecule to physically interact with the target at the same time as the binding molecule. Supramolecules are formed containing at least two supramolecular component molecules that contain an effector molecule and a nucleic acid chain. A nucleic acid chain on a component molecule is complementary to a nucleic acid chain on another component molecule to enable binding of the component molecules of the supramolecule by the formation of double stranded nucleic acid chains between complementary chains. A targetable antiviral supramolecule is prepared containing spectrin as the joining component. The binding molecule can be an antibody specific for an antigen on a viral particle and the therapeutic molecule can be an enzyme capable of destroying infectivity of the virus by hydrolysis of viral coat protein or viral lipid.

RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/424,874, filed Apr. 19, 1995, now U.S. Pat. No. 5,718,915, which is acontinuation-in-part of application Ser. No. 08/332,514, filed Oct. 31,1994, now abandoned.

1. FIELD OF THE INVENTION

The present invention is in the field supramolecular structures thatspecifically bind to a selected target.

2. BACKGROUND

Organized molecular systems are well known in biology and chemistry. Forexample, pure molecular compounds form crystals, and surface activemolecular compounds form monolayers at air-water interphase and vesiclesin water. Bilayers of liposomes mimic biological membranes, andbiological membranes are good examples of multimolecular organizedsystems. Viruses, in particular, are highly organized supramolecularassemblies whose complexity surpasses any man-made assembly. Anotherprime example is the DNA double helix, which is the result of highlyselective interaction of two complementary single strand molecules. Manmade, or artificial examples of supramolecular systems, includecryptates, i.e., inclusion complexes of macrocyclic receptor molecules,and interrupting two dimensional hydrogen bonded network by a largecapping molecule. In these state-of-the-art examples, the structure ofall participating molecules are highly specific.

Jean-Marie Lehn has defined supramolecular chemistry as the chemistrybeyond individual molecules, i.e., the chemistry of the intermolecularbond. Early work in supramolecular chemistry involved crown ethers andcryptates, compounds based on the interaction of electron pair and ionand possibly additional ion-ion interaction (J.-M. Lehn, Angew. Chem.Int. Ed. Engl. 29 (1990) 1304-1319).

Oligobipyridines form in the presence of suitable metal cations such ascopper(II) double-stranded helicates. Auxiliary groups may be attachedinto bipyridine units. If these groups are nucleotides they may serve asrecognition sites for DNA (U. Koert, M. M. Harding and J.-M. Lehn,Nature (1990) 346:339).

Most previously described hydrogen bonded supramolecules aresupramolecular polymers, i.e., periodic supramolecules composed of oneor two repeating units. In principle the number of repeating units ofpolymeric supramolecules may be larger than two but until now nobody hasused more than two repeating units. Examples of this class ofsupramolecules includes the chain-like supramolecule formed byco-crystallization of 1:1 mixture of 2,4,6-triaminopyrimidine and asuitable barbituric acid derivative (J.-M. Lehn, M. Mascal, A. DeCian,J. Fisher, J. Chem. Soc. Chem. Commun. (1990) 479).

Polymeric supramolecules formed from a single unit may also be used. Forexample, a tubular supramolecule has been formed from a single cyclicpeptide (M. R. Ghadiri, J. R. Granja, R. A. Milligan, D. E. McRee and N.Khazanovich , Nature (1993) 366:324-327). These polymeric supramoleculesare often simply crystals or mixed crystals in which hydrogen bondingplays a predominant role in structure maintenance. Even, if thesesupramolecules are stable in solution, their size is variable like thatof a conventional polymer.

A step towards controlling supramolecular size and shape has been theuse of capping molecules to interrupt the molecular association at thedesired point (J. P. Mathias, C. T. Seto, J. A. Zerkowski and G. M.Whitesides in "Molecular Recognition: Chemical and Biochemical ProblemsII" (Ed. S. M. Roberts) Royal Society of Chemistry). A mixture of heisocyanurate derivative (benzCA₂) and trismelamine derivative (trisM₃)gives the supramolecule (trisM₃)₂ (benzCA₂)₃. This strategy typicallyproduces supramolecules which have `molecular weight` of 4-10 KDa.

No process exists today for creating large molecular assemblies ofdeliberately chosen molecules in which the location of the molecules inthe assembly can be selected accurately with respect to each other.Nonetheless, a dire need exists for such molecular structures since theycould have numerous important medical, chemical and physicalapplications. These applications include, but are not limited to,supramolecular drugs, drug delivery to target organs, capture of virusesand catalysts, sensors and nanotechnological components.

Polypeptides and proteins, especially enzymes, have been attached tooligonucleotides. A peptide or protein has been used as a tag for anoligonucleotide or oligonucleotide is used as a tag for a polypeptide.Techniques such as ELISA allowed to trace enzymes easier thanoligonucleotides, enzymes were used as tags for oligonucleotides. PCRprovides for assays of extreme sensitivity. Oligonucleotides are oftenused as a tag for polypeptides or peptidomimetics, so that the fate ofthe polypeptide can be followed in vitro or in vivo. Synthesis methodswhich are used to prepare these conjugates are also useful in thisinvention. (D. Pollard-Knight, Technique (1990) 3:113-132).

Linear single-stranded tRNA forms branched structures because there areseveral complementary pieces of the sequence are suitably located.Recently, several two and three dimensional structures have been formedusing this principle (Y. Zhang and N. C. Seeman, J. Am. Chem. (1994)116:1661-1669; N. C. Seeman, J. Theor. Biol. (1982) 99:237-247.). TheseDNA based supramolecules have been bound together to form activestructures. Because several steps are typically needed to create thesemolecules, the overall synthesis yield can be very low (0.1-1') becauseof these steps alone.

Branched pre-mRNA is found in cells. These molecules have highlyspecific structures in which adenosine is always linked to guanosine.These branched RNAs have been synthesized (T. Horn and M. S. UrdeaNucleic Acid. Res. (1989) 17:6959-6967; C. Sund, A. Foldesi, S.-I.Yamakage and J. Chattopahyaya, Nucleic Acid. Res. (1991) 9-12). Thesynthesis of branched nucleic acids has been extended to the synthesisof nucleic acid dendrimers (R. H. E. Hudson and M. J. Damha, J. Am.Chem. Soc. (1993) 113:2119-2124).

Oligonucleotide comb and fork structures have been used for analyticalpurposes (M. S. Urdea, B. Warner, J. A. Running, J. A. Kolberg, J. M.Clyne, R. Sanchez-Pescador and T. Horn (Chiron Corp.) PCT Int. Appl. No.WO 89/03,891 05 May 1989, U.S. application Ser. No. 109,282, Oct. 15,1987. 112 pp).

All previously known supramolecular structures have some drawbacks. Itis of interest to provide novel supramolecular structures that may beadapted for a variety of uses, including disease therapy, diagnostics,assays, and electronics.

3. SUMMARY OF THE INVENTION

The present invention provides several different bindingmolecule-multienzyme complexes capable of specifically binding to atarget of interest. The binding molecule-multienzyme complexes of theinvention comprise two or more different effector molecules joined toeach other by a joining component, wherein at least one of the effectormolecules has the property of binding to a molecular target, i.e. abinding effector molecule, and at least one of the other effectormolecules is a therapeutic effector molecule. The joining components foruse in the binding molecule-multienzyme complexes of the invention maybe of a variety of classes including liposomes, proteins, organicpolymers (including dendrimer type polymers). Another aspect of theinvention to provide binding molecule-multienzyme complexes in which thejoining component is of sufficient length and/or flexibility to permitthe therapeutic effector molecules to physically interact with the sametarget as binding molecule at the same time as binding effector moleculeis interacting with the target.

One aspect of the invention relates to binding molecule-multienzymecomplexes that are supramolecules formed by at least two supramolecularcomponent molecules. Each supramolecular component molecule comprises atleast one effector molecule and at least one nucleic acid chain. Atleast one of the nucleic acid chains on at least one component moleculeof the supramolecules of the invention are complementary to nucleic acidchains on at least one other component, and thus are able to bind thecomponents of the supramolecule by the formation of double strandednucleic acid chains between the complementary chains. The presentinvention also provides methods of making the supramolecules of thepresent invention.

The nucleic acid chains of the supramolecules of the invention arepreferably DNA, RNA and may also contain structural analogues of DNA orRNA. Effector molecules that may be used to form the supramoleculesinclude, but are not limited to polypeptides, proteins, lipids, sugars.These effector molecules may impart chemical and physical properties tothe supramolecule include, hydrophobicity, hydrophilicity, electronconductivity, fluorescence, radioactivity, biological activity, cellulartoxicity, catalytic activity, molecular and cellular recognition and invivo transport selectivity.

Another aspect of the invention is to provide bindingmolecule-multienzyme complexes of the invention that may be used totreat or prevent infectious diseases, particularly viral infectiousdiseases. Binding molecule-multienzyme complexes suitable for thetreatment and/or prevention of infectious diseases comprise effectormolecules that are antibodies specific for one or more antigen on aviral particle and one or more enzyme capable of catalyzing a reactionthat destroys the infectivity of the virus of interest, e.g., hydrolysisof viral coat proteins or viral envelope lipids.

An effector molecule for use in the invention may also be a toxin, suchas ricin, which will kill the cell, if the virus is internalized.Another aspect of the invention is to provide bindingmolecule-multienzyme complexes adapted for the treatment ofnon-infectious diseases. Binding molecule-multienzyme complexes for thetreatment of specific diseases may comprise binding effector moleculesspecific for certain cells or tissues and effector molecules that servesto directly alleviate a given disease condition.

Another aspect of the invention is to provide bindingmolecule-multienzyme complexes that expedite the delivery ofpolynucleotides and other macromolecules into the interior of cells.Such binding molecule-multienzyme complexes are supramolecularstructures derived from two or more supramolecular components adaptedfor the internalization of macromolecules may comprise effectormolecules that either alone, or in combination with other effectormolecules, on the same or different structure, that are capable ofcrosslinking receptors on the surface of a cell for transformation.

Another aspect of the invention is to provide bindingmolecule-multienzyme complexes useful for performing assays forcompounds of interest, particularly immunoassays. Supermolecularstructures for use in assays typically comprise an effector moleculecapable of specifically binding to a compound of interest and a secondeffector molecule that may capable of producing a detectable signal,e.g., an enzyme, or a second molecule capable of specifically binding toa compound of interest. Another aspect of the invention is to provideassays employing binding molecule-multienzyme complexes of the inventionso as provide for the detection and/or quantitation of compounds ofinterest.

Another aspect of the invention is to provide bindingmolecule-multienzyme complexes useful for the prevention and treatmentof atherosclerosis and related cardiovascular disorders. Bindingmolecule-multienzyme complexes of the invention useful for the treatmentof such diseases may comprise an effector molecule that is an antibodyspecific for antigens in atherosclerotic plaque.

Another aspect of the invention is to provide method and compositionsfor the genetic manipulation of cells of interest. The methods andcompositions may be used for in vivo and in vivo genetic therapy. Thecompositions for use in genetic therapy are binding molecule-multienzymecomplexes adapted for genetic manipulation. Preferably, such bindingmolecule-multienzyme complexes adapted for genetic therapy compriseliposomes as joining molecules and further comprise a geneticmanipulation complex. Genetic manipulation complexes comprise apolynucleotide for genetic manipulation, a motor protein, and aplurality of site-specific DNA binding proteins. Preferably, the geneticmanipulation complex comprises and enzyme with phospholipase activity.

4. BRIEF DESCRIPTION OF THE FIGURES

The invention will be better understood by reference to the appendedFigures, of which:

FIG. 1 is a schematic representation of the construction of asupramolecule constructed from two components.

FIG. 2(A) is a schematic representation of the construction of a squareplanar supramolecule constructed from four components.

FIG. 2(B) is a schematic representation of the construction of a squareplanar supramolecule constructed from four components which isreenforced by diagonal double stranded nucleic acid chains.

FIG. 2(C) is a schematic representation of the construction of atetrahedral supramolecule constructed from four components.

FIG. 3(A) is a schematic representation of the construction of anantibody-multienzyme supramolecule constructed from supramolecularsubcomponents.

FIG. 3(B) is a schematic representation of the construction ofsupramolecular subcomponents used in FIG. 3(A) from molecules eachcontaining one enzyme or antibody.

FIG. 3(C) is a schematic representation of the construction of twosupramolecules containing an antibody and two enzymes. The combinationof these two supramolecules is able to degrade all lipid components ofthe virus.

FIG. 4 is a schematic representation of a supramolecule subcomponentwhich is capable of forming a supramolecular cage around a virus when itcombines with a complementary supramolecule subcomponent.

FIG. 5A is a schematic representation of a typical icosahedral virus.

FIG. 5B is a schematic representation of the supramolecule subcomponentof FIG. 4 approaching the icosahedral virus.

FIG. 5C depicts a second, complementary supramolecule subcomponentapproaching the icosahedral virus.

FIG. 5D depicts two complementary supramolecule subcomponentssurrounding a icosahedral virus.

FIG. 5E depicts a icosahedral virus encased within a supramolecule.

FIG. 6 is a schematic representation of how the analogous structure forthe large molecule in FIG. 4 can be prepared using smaller molecules.

FIG. 7 is a schematic representation of molecules needed to constructthe supramolecule of FIG. 5.

FIG. 8 is a schematic representation of supramolecular assemblies whichare similar to the molecules shown in FIG. 6.

FIG. 9 is a schematic representation of the use of triple helices insupramolecular assemblies.

FIG. 10 illustrates an example of a spacer molecule for connecting threenucleotides to an effector molecule.

FIG. 11 illustrates an example of a second spacer molecule forconnecting three nucleotides to an effector molecule.

FIG. 12 illustrates an example of a method for cross-linking twocomplementary oligonucleotides at one end.

FIG. 13 illustrates an example of the coupling of two derivatizedpeptide chains to form a branched peptide structure which can serve as atrivalent linker.

FIG. 14 illustrates an example of a method of using the manipulation ofprotective groups on a trivalent spacer in order to use the trivalentspacer in oligonucleotide synthesis.

FIG. 15A is a schematic representation of a supramolecule adapted fortransformation of a nucleic acid of interest into a eukaryotic cell.

FIG. 15B is a schematic representation of a supramolecule adapted fortransformation into a cell.

FIG. 15C is a schematic representation of a supramolecule adapted fortransforming a cell and the internalization, i.e., transformationprocess.

FIG. 16 is a schematic diagram of proteinaceous effector moleculecovalently coupled to a phospholipid which may be incorporated into thebinding molecule-multienzyme complexes of the invention.

FIG. 17 is a schematic diagram of liposomal binding molecule-multienzymecomplex of the invention adapted for use in treating atherosclerosis.The liposomal binding molecule-multienzyme complex comprises antibodiesan binding effector molecules and lipase, urokinase, phospholipase A₂,and cholesterol esterase as therapeutic effector molecules.

FIG. 18 is a schematic diagram of liposomal binding molecule-multienzymecomplex of the invention adapted for use in treating HIV-1 infectionsand the interaction of the complex within an HIV-1 virion. Diffusion ofthe effector molecules in the liposome are demonstrated. The symbol "A"refers to phospholipase A₂ used as a therapeutic effector molecule. Thesymbol "C" refers to cholesterol esterase used as a therapeutic effectormolecule. The symbol "L" refers to lipase used as a therapeutic effectormolecule.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to composition of matter that have theproperty of specifically binding to selected molecular targets ofinterest, and directing a therapeutic agent to the binding site. Thecompounds of the invention are collectively referred to as bindingmolecule-multienzyme complexes of the invention comprise two or moredifferent effector molecules joined to each other by a joiningcomponent, wherein at least one of the effector molecules is a bindingeffector molecule and at least one of the effectors is a therapeuticeffector. The joining component may be any of a variety of formsincluding liposome, proteins, organic polymers, and the like.

One of the types of binding molecule-multienzyme complexes of theinvention are supramolecules (also referred to herein as supramolecularassemblies and supramolecular constructions) that comprise at least twocomponents, i.e., supramolecular components. Each supramolecularcomponent comprises an effector molecule and at least one nucleic acidchain covalently joined to the effector molecule. By placingcomplementary nucleic acid chains on different components, thecomponents of the supramolecule may be bound together by the associativeforces, i.e., hydrogen bonding, between the complementary nucleic acidchains, thereby producing supramolecular constructions in which two ormore effector molecules are joined to one another a double-stranded orpartially double stranded nucleic acids.

The general concept of the supramolecular assembly embodiment of theinvention may be better understood by reference to FIG. 1 whereinsupramolecular components A and B ar joined by effector molecules M andN, respectively. Components A and B are bound to each other by thedouble stranded nucleic acid chain formed by complementary nucleic acidchains.

There is no theoretical limit to the number of supramolecular componentsthat may be used to construct a particular supramolecular assembly.Rather, steric factors that could limit the number of components thatcan be used in a particular supramolecule may be avoided by properdesign of the supramolecule using basic structural information that iswell known to the person of ordinary skill in the art of biochemistry.Thus the invention provides for numerous compounds that aresupramolecular assemblies, i.e, supramolecules, comprising two or moresupramolecular components of the invention. The supramolecularcomponents of the invention comprise an effector molecule, e.g., anantibody, covalently joined to at least one polynucleotide. Two or moresupramolecular components of the invention may be joined to one anotherby means of the nucleic acids moieties of the supramolecular componentsby employing nucleic acids that have regions of complementarily orpartial complementarily to one another. Thus two or more effectormolecules may be joined to one another by double stranded or partiallydouble stranded nucleic acids.

Any class of molecule can be used as effector molecule component of thesubject binding molecule-multienzyme complexes. Suitable molecules foruse as the effector molecule moieties of the subject bindingmolecule-multienzyme complexes include, but are not limited to, sugars,peptides, lipids, polymers. The effector molecules of the bindingmolecule-multienzyme complexes may serve several different functionswithin the binding molecule-multienzyme complexes. For example, theeffector molecules may be used to provide a wide array of structuralfeatures to binding molecule-multienzyme complexes. In addition, theeffector molecules can also provide certain chemical and physicalproperties to the binding molecule-multienzyme complexes, which include,but are not limited to, hydrophobicity, hydrophilicity, electronconductivity, fluorescence, radioactivity, biological activity, cellulartoxicity, catalytic activity, as well as molecular and cellularrecognition and in vivo transport selectivity. Effector moleculesinclude a variety of protein type, including toxins, proteinases,receptors, ligands, lectins, antibodies, esterases, hormones, cellsurface markers, etc.

Some effector molecules are referred to herein as "binding effector"molecules. Binding effector molecules have the property of specificallybinding to one or more molecules of interest, i.e., target molecules. Agiven binding effector molecule has the property of being a bindingeffector molecule with respect to target molecules. Effector bindingmolecules may be either a member of a pair of specific bindingmolecules, e.g., an antibody-hapten pair or a receptor-ligand pair.Examples of binding effector molecules include, but are not limited to,antibodies, integrins, adhesins, cell surface markers, T cell receptors,MHC proteins, and the like. By using a combination of two or moredifferent binding effectors, each having a binding specificity for adifferent target, greater binding specificity of the bindingmolecule-multienzyme complexes of the invention may be achieved.Combinations of two or more different binding effector molecules may beused in embodiment of the invention that employ liposomes as joiningcomponents as well as other molecules as joining components.

Suitable binding effector molecules include molecules capable ofspecifically binding to the constant region of antibodies. Theseantibody-specific binding molecules may be antibodies. Such antibodyspecific antibodies are well known in the art and are commerciallyavailable from many sources, e.g. Sigma Biochemical (St. Louis, Mo.).Additionally, the antibody-specific binding molecules may be moleculesother than antibodies; for example, macrophages and other leukocyte haveFc receptor proteins on their surfaces. These Fc receptors may be usedas antibody-specific binding molecules so as to function as bindingeffector molecules. As endogenously produced antibodies may accumulateat the site or sites of infections and/or tumors, thereby directingbinding molecule-multienzyme complexes.

Another class of effector molecules are referred to herein as"therapeutic effector molecules". Therapeutic effector molecules areeither (1) biologically active molecules, including but not limited to,enzymes, drugs, prodrugs, enzymes, ligands specific for receptors,radionuclides, and toxins or (2) detectable labels, including, but notlimited to fluorophores, enzymes, and radionuclides. A given therapeuticeffector molecule may be both a biologically active molecule anddetectable label. The following are some examples of therapeuticeffector molecules that have been used to treat various diseases. Thesetherapeutic effector molecules may readily be adapted for use in theclaimed invention. Thrombolytic therapy is well established in thetreatment of acute myocardial infarction (Z. Bode, et. al., Z. Kardiol83 (1994) 393). Proteinases which have been approved for this purposeinclude urokinase and streptokinase. Recombinant plasminogen activator,which activates plasminogen occurring naturally in the blood, is also incommon use. Proteolytic enzymes have been also used to treat diskherniation (chymopapain, L. G. Lenke, et. al., AORN J. 59 (1994) 1230),fallopian tube (chymotrypsin, chinese) and inflammation(carboxypeptidase N, M. Rybak et. al. Pharmacology 16 (1978) 11). Otherenzyme therapies studied include: Adenosine deaminase, DNAse (cysticfibrosis), Glucosylceramidase (Gaucher's disease), Lipase (cysticfibrosis, G. Morrison et. al., Aliment Pharmacol. Ther. 6 (1992) 549;cancer B. A. Richards, J. R. Soc. Med. 81 (1988) 284), Lysozyme(immunostimulant; antibacterial agent, I. A. vereshchagin and O. D.Zhuravleva, Vrach. Delo (1994) 103), Peptide hydrolases (antiviralagents, K. N. Veremeenko, Vrach. Delo (1994) 8), Superoxide dismutase,Terrilytin (immunostimulant), Ribonuclease (antibacterial, antiviral,antitumor), Phospholipase A2 (autoimmune suppression, K. Mahlberg, et.al., Acta Ophthalmol. Suppl. 182 (1987) 166); arthritis, R. B. Zurier,et. al., Ann. Rheum. Dis. 32 (1973) 466), Phospholipase C (pulmonarymicroembolism, I. G. Jansson, et. al., J. Trauma 28 (1988) S 222.

The binding molecule multienzyme complexes of the invention comprise atleast one binding effector molecule and at least one therapeuticeffector molecule. The binding molecule-multienzyme complexes maycomprise more than one different type of binding effector molecule andmay contain more than one molecule of each type of binding effectormolecule. Similarly, the binding molecule-multienzyme complexes of theinvention may comprise more than one different type of therapeuticeffector molecule and may contain more than one molecule of each type oftherapeutic effector molecule present in the effector molecule. Thespecific effector molecules within a given binding molecule-multienzymecomplexes may be selected so that the target component molecule hastherapeutic value for a given disease or diseases. The person ofordinary skill in the art, given the information provided herein, willreadily be able to select appropriate effector molecules to treat adisease of interest. For example, a target multi-component therapeuticmolecule for the treatment of a viral disease may comprise an antibodyspecific for a viral coat protein or viral envelope lipid as a bindingeffector molecule and a proteinase specific for the same viral coatprotein as a therapeutic effector molecule.

The nucleic acid used to join the subject supramolecular components toeach other in some embodiments of the invention, i.e., targetedmulti-component supramolecules comprising two or more supramolecularcomponents, are preferably between 5 and 100 bases in length, althoughnucleic acids may be significantly longer than 100 bases. The nucleicacid portion of the subject supramolecular components and supramolecularassemblies may be any of the wide variety nucleic acid molecules, eithernaturally occurring, e.g., RNA or DNA, or synthetic analogs, e.g.,phosphorothioates. The term "nucleic acids" as used herein, unlessindicated otherwise, refers to both naturally occurring nucleic acidsand synthetic analogs thereof. For many applications, it may bedesirable to use synthetic analogs of natural nucleic acid rather thannucleic acids because of certain properties specific to the analogse.g., nuclease resistance and higher denaturation temperatures ofdouble-stranded nucleic acids.

Detailed descriptions on the use and synthesis of nucleic acid analogscan be found, among other places, in U.S. Pat. No. 5,292,875(phosphorothioates), U.S. Pat. No. 5,218,103 (thiophosphoramidites),U.S. Pat. No. 5,183,885 (phosphorothioates), U.S. Pat. No. 5,151,510(phosphorothioates), U.S. Pat. No. 4,814,448 (phosphonates), U.S. Pat.No. 4,096,210 (phosphorates) U.S. Pat. No. 4,094,873(phenylphosphorothioates), Ragle et al., Nuc. Acids. Res. 18(6):4751-4757 (1990) (phosphoramidates). Information on how to synthesizeconventional nucleic acid can be found, among other places, in EcksteinOligonucleotide and Analogues: A Practical Approach Oxford UniversityPress (1992). The complementary nucleic acids need not necessarily beentirely complementary with respect to one another. A nucleic acid of afirst supramolecular component may be complementary to only a portion ofthe nucleic acid moiety of a second supramolecular component or thecomplementarity may be over the entire length of the nucleic acid.Nucleic acid moieties of the subject supramolecular components maycontain multiple regions of complementarily to two or more nucleic acidsmoieties on additional supramolecular components thereby to be joined topermitting three or more supramolecular components to be joined to oneanother through hybridization. The complementarily (as measured bysequence homology) may be either 100 percent or less. It will beappreciated by those of ordinary skill in the art that the strength ofassociating, as indicated by duplex nucleic acid melting point, may bemodulated by controlling factors such as the degree of complementarily,the identity of the base pairs (e.g., GC rich nucleic acids have ahigher Tm than AT rich nucleic acids), the choice of a nucleic acid ornucleic acid analog, the length of the region of complementarily, andthe like. The nucleic acid moieties of the subject supramolecularcomponents may be linear or branched. Methods of producing branchednucleic acids are known to the person skilled in the art, and example ofhow to make branched nucleic acid molecules can be found in PCTPublication No. WO 89/03891. The use of branched nucleic acids as thenucleic acids as the nucleic acid moieties of the subject supramolecularcomponents is particular interest because branched nucleic acid may beused to conveniently join three or more supramolecules components to oneanother through hybridization of the nucleic moieties. Triple and tetrahelixes of nucleic acid chains can also be used in the supramolecules inorder to provide other structural characteristics, such as rigidity, tothe supramolecule.

The length of the nucleic acid moieties as well as the position of thecomplementary base on the nucleic acids may be used to control the twoand three dimensional shape of the supramolecule. For example, asdepicted in FIG. 2(A), a square supramolecule can be prepared byemploying four components which each contain two nucleic acid chains ofequal length. Similarly, as also depicted in FIG. 2(C), a tetrahedralsupramolecule can be formed using four components. As can be seen fromFIGS. 2(A) and 2(C), a wide variety of two and three dimensionalsupramolecule structures may be formed using differing numbers ofcomponents and differing numbers of complementary nucleic acid chains.For example, supramolecules of the present invention may containgeometric configurations that generally resemble triangles, squares,pentagons, hexagons, heptagons, octagons, parallelograms, pyramids,tetrahedrons, cubes and cylinders. It should also be understood thatthese figures are merely schematic representations of supramolecularassemblies and that the supramolecule may not actually possess thesegeometric structures in solution or in crystalline form because of thedue to the flexibility of double stranded nucleic acid chains as well asother salvation, electronic and stearic factors that may be present in agiven supramolecule.

With respect to each supramolecular component, the number of nucleicacid moieties that may be attached to a particular effector molecule maybe varied greatly so as to produce supramolecular assemblies of thedesired structure. Supramolecular components of the invention maycomprise one or more nucleic acid moieties. The total number of nucleicacid moieties that may be attached to an effector molecule is limited bystearic hinderance and the number of potential attachment sites,problems which may be avoided by proper selection of the effectormolecule and the nucleic acid moieties.

In another embodiment of the supramolecular components of the invention,more than one effector molecules may be joined to a single nucleic acidmolecule. Such supramolecular components comprising a plurality ofeffector molecules joined to a single nucleic acid molecule may be usedto form supramolecular assemblies through a nucleic acid hybridizationwith the nucleic acid moieties of similar supramolecular components orsupramolecular components in which nucleic acid moieties are joined toonly a single effector molecule.

In a preferred embodiment of the binding molecule-multienzyme complexesof the invention the individual effector molecules are joined to oneanother by a joining component (or joining means) that gives theindividual effector molecules within the binding molecule-multienzymecomplexes operational freedom, i.e., freedom of movement sufficient topermit a therapeutic effector component to physically interact with thesame target as a binding effector component of the same bindingmolecule-multienzyme complexes when the binding effector component isbound to the target. Several different types of molecules andmulti-molecular assemblies may serve as joining components in differentembodiments of the invention. The embodiment of the invention in whichthe various effector molecules are interconnected by joining componentsthat permit the effector molecules to have the aforementioned degree ofoperational freedom are not limited by the examples provided herein. Theperson of ordinary skill in the art of chemistry and/or molecularbiology will be able to devise numerous joining components having thedesired characteristics. For example conventional bifunctional linkermolecules may be modified a person of ordinary skill in the art organicchemistry go as have sufficient length to function in the joining ofeffector molecules with operational freedom. In one embodiment of theinvention, the joining component is a liposome, i.e. the bindingmolecule-multienzyme complexes is a liposome comprising a target bindingmolecule and a therapeutic effector molecule. In another embodiment ofthe invention, the joining component is a peptide, i.e. bindingmolecule-multienzyme complexes comprises a binding effector molecule anda therapeutic effector molecule joined to each other through a peptide.In another embodiment of the invention, the joining component is adendrimer, i.e., binding molecule-multiemzyme complexes is a dendrimercomprising a binding effector molecule and a therapeutic effectormolecule.

When the binding molecule-multienzyme complexes of the inventioncomprises a liposome. The effector molecules are covalently attached toone or more constituent lipids of the liposome. The effector moleculemay be directly coupled to the lipid, or coupled through a linker.Linkers serving to couple an effector molecule to a lipid may of avariety of compounds, including polynucleotides. The liposome mayoptionally comprise one or more therapeutic effector molecules in theinternal compartment of the liposome as well as covalently attached tothe surface of the liposome. Liposomes can be classified as smallunilamellar vesicles, large unilamellar vesicles, cell-size unilamellarvesicles and multilamellar vesicles (J. M. Wrigglesworth in MembraneProcesses, Molecular Biology and Medical Applications (Eds. G. Benga, H.Baum and F. A. Kummerow), Springer-Verlag, New York, 1984). Methods ofpreparation of these liposomes include vortexing, sonication, detergentdialysis or dilution, infusion or reverse phase evaporation, fusionmethods, addition of short-chain PC's, addition of fatty acids ordetergents, rapid extrusion and transient increase in pH.

The simplest way to prepare liposomes is to add water onto the drycomponents, and mix. The power used in the mixing determines the size ofthe liposomes. Other possibility is to use small molecular weightdetergents to solubilize the lipid-protein components and phospholipids.The detergent is dialyzed off the solution and lipooomes formspontaneously. Also the pure lipid liposomes can be prepared in acontrolled manner and lipid-protein conjugates may be added later in adetergent solution. Lipid part of the conjugate will penetrate into thebilayer and the proteins will be anchored. The detergent can be dialyzedaway. This approach uses relatively small amount of detergent, which canbe biocompatible, and need not be removed.

The lipid part of a liposome that is coupled to an effector moleculethat is a protein preferably contains primary amino or thiol group intheir polar head group. Accordingly, phosphatidylethanolamines can bedirectly used. Thiol groups can be introduced into syntheticphospholipids, although it is not a part of natural lipids. In order toanchor the lipid-protein conjugate firmly into the liposome thelipophilicity of the lipid should be increased. This can be achieved inseveral ways. First, the length of the alkyl chains in the lipid can besignificantly increased (about C₃₀) compared what is normally used(about C₁₈) in liposomes. Secondly, the number of alkyl chains in onelipid molecule can be higher than two, e.g., five alkyl chainphospholipid can be conveniently prepared starting from mannitol.Alternatively, several phospholipid molecules can be chemically coupledthrough their polar head groups into a controlled multimer, which isfurther conjugated with a protein. Thirdly, phospholipids that arethermophilic bacterial phospholipids, or an analog thereof can be used.These phospholipids are formally like phospholipid dimers, in which thetwo phospholipids are connected via their alkyl tails. Either one orboth alkyl tails can be connected. Thus, a monomolecular layer in aliposome may look like a bilayer formed by a normal phospholipid. Thestability of the liposomes formed by these `dimeric` phospholipids isvery high. Fourthly, polymerizable phospholipids can be used. Thepolymer is preferably biocompatible and biodegradable, preferably apolyester or a polyamide.

The lipid component of the embodiments of the subject bindingmolecule-multienzyme complexes that are liposomes are preferably notsubstrates of lipolytic enzymes used as effector molecules. When lipaseand phospholipase A₂ are used as an effector molecule, the lipids mustbe resistant against these enzymes. If ether bonds are used to connectalkyl chains with glycerol or other polyalcohols, the resistance isautomatically attained, because these two enzymes are ester hydrolases.Enzymes are also sensitive for the stereochemistry of the lipid. Use ofunnatural stereochemistry reduces or completely inhibits enzymaticactivity. The liposomal lipid bilayer can be highly analogous to lipidpart of cellular membranes. The liposomes of this invention may alsocomprise proteins in addition to the effector molecules. These proteinsare not normal membrane proteins and their concentration in the blood isvery low. In order to make these liposomes to resemble normal bloodcomponent or cell, additional proteins may be attached onto theirsurfaces. Possible proteins include albumin and glycophorin. Albumin isa molecular level scavenger, which removes fatty acids and lysolipids.These lipids are formed via the action of the lipolytic enzymes, whichare a part of the liposome. Thus, albumin is an ideal additionalcomponent of the liposome, because it can perform a double function:camouflage and scavenging the hydrolytic products. Glycophorin is amembrane bound glycoprotein. It will make these liposomes to look likecells to some degree.

An advantage of the liposomes is that the number of effector moleculescan be easily adjusted without affecting the size of the liposome. Thevarious components may be supposed to be randomly distributed over thesurface. Because of the rapid diffusion, the effector molecules whichare needed to perform a certain function, are available when needed.Once binding effector molecules makes a contact with the target, otheradhesion molecules will diffuse to the vicinity of the contact site andwill bind to the target. Most targets have several binding sites neareach other. The strong binding between binding effector molecules andtheir targets is preferred, but is not necessary for the liposomescontaining digestive enzymes. For instance, by removing the lipid coatof the HIV-1, the protein core of the virus will dissolve and RNA isexposed. This happens when the virus is internalized by the cell. Thelipid bilayer of the virus will fuse with the cellular membrane and theRNA is released. This is in contrast to influenza virus, which has anendocytosis pathway. The infected cell dismantles the influenza virus inlysosomes using lipases and proteases. For HIV-1 proteases are notneeded. Thus, proteases are not required needed to inactivate HIV-1 andsimilar viruses, while for influenza virus and other viruses with asimilar life cycle, proteases may be effective for inactivating thevirus. The liposome internal compartment may also comprise an antisenseoligonucleotide or other therapeutic compound such as a drug or enzyme,In some embodiments of the invention, the liposomes may comprise a DNAseor RNAse in the internal compartment of the liposome or as therapeuticeffector molecules on the surface of the liposome.

Many naturally occurring proteins may be used without any modificationas effector molecules. Because in some cases these liposomes are used invivo in humans, the human proteins should be used to avoid unwantedimmune responses. Human proteins can be produced in transgenic plants oranimals. The amino acid sequence of proteins may also be altered throughwell-known genetic engineering techniques to produce mutated proteinshaving the desired biological functions of corresponding naturallyoccurring protein, but adapted to coupling to lipid molecules. Forexample, addition of a cysteine residue, either through substitution orinserting, will add a free thiol group for coupling to a lipid molecule.Moreover the location of this cysteine can be deliberately chosen. Itshould be located so that conjugation does not disturb the activity ofthe protein. Normally this means that the cysteine must be as far aspossible from the active site.

In order to covalently couple a lipid with an effector molecule, thelipid must contain a functional group which has high enough reactivitywith heterobifunctional cross-linker. Phosphatidylethanol-amines (PE)have free aliphatic amino group and can be used directly forconjugation. The conjugation of phosphatidylethanolamines (PE) andserines (PS) with proteins is well known in the literature (Egger et.al. Biochem. Biophys. Acta 1104 (1992) 45-54). Especially, if theprotein contains a thiol group, a chemically well defined conjugate canbe easily prepared. Several spacers are commercially available to coupletwo molecules, which contain an amino and a thiol group. If a proteindoes not contain an a thiol group, an aliphatic amino group, may be usedfor conjugation. Thiol groups are preferred for conjugation sites inproteins as compared with amino groups because of the lower abundance ofthiol groups in most proteins.

Proteins can also be conjugated with lipids after the preparation of theliposomes so as to position the proteins on the outer surface of theliposome. Phospholipids having unnatural stereochemistry may be preparedas described in the literature, for example see, (J. A. Virtanen et.al., Chem. Phys. Lipids 27 (1980) 185). D-Mannitol is tritylated toyield 1,6-ditrityl-D-mannitol, which is oxidized with lead tetra-acetateand the product reduced with sodium borohydride to yield1-trityl-sn-glycerol (1-TrG). Diacylglycerols and phospholipids can beprepared starting from 1-TrG by standard methods. For the liposomesdescribed in this application ether bond is preferred at least in theprimary hydroxyl group, because it is resistant against lipase. In someembodiments an sn-hydroxyl ester bond may be useful because theunnatural stereochemistry makes this bond stable against phospholipaseA₂. Other lipids for use in the subject liposomes include2-oleoyl-3-triacontanyl-sn-glycero-1-phosphatidyl ethanolamine and other2-acyl-2-alkyl-PE's.

In another embodiment of the binding molecule-multienzyme complexes ofthe invention, in which the joining component is a peptide. The peptidemay be either naturally occurring, e.g., spectrin or fibrin, or may beartificial. The peptide joining component may take the form of a largefusion protein when the binding effector molecule and the therapeuticeffector molecule are both proteins. One advantage of using bindingmolecule-multienzyme complexes that are fusion proteins is that thecompound may be produced by standard recombinant DNA productiontechniques such as those known to the person of ordinary skill in theart, such as described in Goedolel, Gene Expression Technology Methodsin Enzymoloqy, Vol. 85 Academic Press, San Diego (1988). In otherembodiments of the invention peptide joining components may be used inwhich the compound is not a fusion protein.

In other embodiments of the invention, the joining component may be adendrimer. A dendrimer is a highly branched polymer. Dendrimers providenumerous sites of attachment for effector molecules at the termini ofthe dendrimer. Information on how to synthesize various dendrimerssuitable for use in conjunction with this invention are well known tothe person of ordinary skill in the art of organic chemistry. Enzymesmay be conjugated with polymers to extend their half-life in the blood(R. F. Sherwood et. al., Biochem J. 164 (1977) 461); M. J. Knauf, et.al., J. Biol. Chem. 263 (1988) 15064; A. Abuchowski and F. Davis in"Enzymes as Drugs" (Eds. J. Holsenberg and J. Roberts) John Wiley andSons, New York, 1981, pp. 367-383). Description of how to join effectormolecules to polymers can be found in R. Labeque, et. al., Proc. NatlAcad. Sci. USA 90 (1993) 3476 (Phospholipase A2 for hypercholesterolemiatreatment). The structure of the polymer is not well defined and thecoupling is even less defined. Only average number of the enzymemolecules per enzyme is known. The coupling of the enzyme occurtypically via lysine residues and because most enzymes contain severallysines, the polymer can be coupled to any of these and possible toseveral lysines in the same enzyme.

The effector molecules can be chemically coupled with a polymer, whichcan be an artificial or a biopolymer. Craft polymers give the highestoperational freedom. Dendrimers having chemically different branches canprovide chemically well defined structure.

The binding molecule-multienzyme complexes of the invention may beproduced in a variety of environments, either in vitro or in vivo.Binding molecule-multienzyme complexes may be constructed in vitro bymixing the various constituents under conditions. Conditions in the invitro reaction mixture may be varied so as to influence the rate ofbinding molecule-multienzyme complexes formation and the nature of thebinding molecule-multienzyme complexes produced.

The binding molecule-multienzyme complexes of the present invention maybe used in a very wide variety of applications which include, but arenot limited to treatment of infectious disease, including HIV-1infections, treatment of atherosclerosis, treatment of cancer,immunoassays, self-assembling resist materials, for electronicself-assembling nanocircuitry, catalytic clusters, sensors,supramolecular drugs, which are capable of encapsulating viruses and/ordestroying viruses. Drug and enzyme targeting to cells and viruses maybe enormously improved by using supramolecular assemblies of theinvention comprising many similar or different monoclonal antibodies andseveral drug molecules, enzymes or other effector molecules.

It will be appreciated by the person of ordinary skill in the art thatthe therapeutic embodiments of the binding molecule-multienzymecomplexes of the invention (e.g., compound for the treatment of cancer,viral infections, atherosclerosis) also include compounds in whicheffector molecules are joined to one another through conventional, i.e.,non-polynucleotide, linkers. The use of non-polynucleotide linkers iswell known the person of ordinary skill in the art and is described in,among other places, in several volumes of the series Methods inEnzymology, Academic Press, San Diego Calif. Examples of suchnon-polynucleotide linkers include, 4-benzoylbenzoic AcidN-hydroxysuccinimide esters, 3-maleimidobenzoic acidN-hydroxysuccinimide esters, 1,4-phenyleneisothiocyanates, and the like.In those embodiments of the invention in which non-polynucleotidelinkers are used to join effector molecules, it may be advantageous toadminister a mixture of different effector molecule conjugates to apatient rather than a large supramolecule. In the treatment of HIV-1infection for example, rather than administer a single supramoleculecomprising (i) an anti-gp120 macromolecule, (ii) a phospholipase, and(iii) a proteinase, it may be desirable to administer a formulationcomprising (i) an anti-gp120-phospholipase conjugate and (ii) ananti-gp120-protease conjugate.

When the binding molecule-multienzyme complexes of the invention areused in vivo, the compounds are typically administered in a compositioncomprising a pharmaceutical carrier. A pharmaceutical carrier can be anycompatible, non-toxic substance suitable for delivery of the therapeuticproteins and nucleic acids to the patient. Sterile water, alcohol, fats,waxes, and inert solids may be included in the carrier. Pharmaceuticallyaccepted adjuvants (buffering agents, dispersing agent) may also beincorporated into the pharmaceutical composition.

The binding molecule-multienzyme complexes components of the inventionmay be administered to a patient in a variety of ways. Preferably, thepharmaceutical compositions may be administered parenterally, i.e.,subcutaneously, intramuscularly or intravenously. Thus, this inventionprovides compositions for parenteral administration which comprise asolution of the human monoclonal antibody or a cocktail thereofdissolved in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers can be used, e.g., water, buffered water,0.4% saline, 0.3% glycerine and the like. These solutions are sterileand generally free of particulate matter. These compositions may besterilized by conventional, well known sterilization techniques. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjusting agents and thelike, for example sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate, etc. The concentration of antibody inthese formulations can vary widely, e.g., from less than about 0.5%,usually at or at least about 1% to as much as 15 or 20% by weight andwill be selected primarily based on fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

Actual methods for preparing parenterally administrable compositions andadjustments necessary for administration to subjects will be known orapparent to those skilled in the art and are described in more detailin, for example, Remington's Pharmaceutical Science, 15th Ed., MackPublishing Company, Easton, Pa. (1980), which is incorporated herein byreference.

An important use of some of the embodiments of supramolecules of theinvention is a 2-dimensional supramolecular structures on semiconductoror other electrically conductive surfaces so that desired patterns ofself-assembling resist materials may be conveniently formed. Thus, theuse of X-rays and electron beam lithography may be avoided when creatingnanometerscale patterns on the semiconductor surfaces. This capabilitywill make a completely new order of nanoelectronics possible.

A second application of major importance will be self-assemblingnanocircuitry using this technique. Pre-prepared diodes, transistors,capacitors, resistors, etc. and wires can be connected in highlyselective ways to form two or three dimensional electronic entities.Electronically conducting complementary polynucleotide chains may beused when electric contact of the nanocomponents is needed.

Binding molecule-multienzyme complexes, of the invention may also beused in catalytic and sensor applications. Employing the bindingmolecule-multienzyme complexes methodology of the present invention,enzymes may be attached to a surface in an organized fashion in order tocreate desired sequential reaction. With regard to sensor applications,a sensor may be created that contains additional biomolecules or organicmolecules that give a photonic or electrical signal when a molecule ofinterest is the supermolecular assembly sensor.

Binding molecule-multienzyme complexes components of the invention maybe used to provide novel immunoassays and related assays for thedetection of compounds of interest. Immunoassay technology is highlydeveloped and well known to person of ordinary skill in the art, see,for example, Hudson, Practical Immunology 3rd Ed. Oxford Publication(1989), and Catty Antibodies: A Practical Approach Volumes 1 & 2 OxfordUniversity press (1989). Conventional immunoassays typically employantibodies conjugated to enzymes, and/or antibody-antibody conjugates.It will be appreciated by one skilled in the art that many embodimentsof the binding molecule-multienzyme complexes, preferably thesupramolecular assemblies may be substituted for the conventionalantibody conjugates used in conventional assays. Bindingmolecule-multienzyme complexes of the invention useful or assay compriseat least one member of a specific binding pair (e.g., an antibody, wherethe specific binding pair of molecules is an antibody and antigentarget) as an effector molecule portion of a bindingmolecule-multienzyme complexes component. Such bindingmolecule-multienzyme complexes supramolecules may, for example comprise(1) two or more specific binding pair members, e.g., antibodies, (2) anantibody and an enzyme capable of generating a detectable signal, e.g.,alkaline phosphatase.

Numerous advantageous variants of conventional immunoassays are enabledby employing the binding molecule multi-enzyme complexes, preferablysupramolecular assemblies of invention instead of conventional antibodyconjugates because certain embodiments of the subject bindingmolecule-multienzyme complexes may be assembled, disassembled, orreassembled during an assay due to the ability of the double-strandednucleic acid moieties of the assembly to disassociate or removal of theappropriate conditions. For example, (i) a binding molecule-multienzymecomplexes comprising antibody joined by a double stranded nucleic acidmolecule to an enzyme producing a detectable signal may be bound to atarget antigen of interest, (ii) the binding molecule-multienzymecomplexes may then be disassociated so as to release the bindingmolecule-multienzyme complexes comprising the enzyme effector molecule(iii) the bound portion of the binding molecule-multienzyme complexesmay then be used to form a new binding molecule-multienzyme complexeswith a second antibody as an effector molecule, thereby permitting theimmobilization of a second molecule of interest at the same location asthe bound binding molecule-multienzyme complexes portion. A person ofordinary skill in the art will appreciate that the properties thesubject binding molecule-multienzyme complexes structures permit manynew and useful assay procedures to be performed.

The binding molecule-multienzyme complexes of the invention may beadapted so as to prevent or treat various infectious diseases, includingHIV-1, the etiological agent of AIDS. Specific infectious organisms maybe targeted by creating, and administering in an effective amount ofbinding molecule-multienzyme complexes of the invention comprising astherapeutic effector molecules, (1) an antibody specific for molecule onthe infections agent and (2) an enzyme capable of catalyzing themodification of some integral structure of the infectious agent. Forexample, a binding molecule-multienzyme complexes adapted for thecontrol of HIV-1 may comprise an antibody specific for one or more HIV-1virus components, e.g., gp120, and one or more of the following enzymes(1) a phospholipase A₂, (2) a lipase, (3) a cholesterol esterase. Byincluding such enzymes in a binding molecule-multienzyme complexes, thelipid bilayer coat the infectious viral particle that may be destroyed.Additionally, binding molecule-multienzyme complexes of the inventionadapted for the treatment of the infectious disease may further compriseof protease capable of degrading a protein component of the infectiousagent and/or a single stranded nucleic acid capable of hybridizing to aportion of the genome of the infectious organism of interest. In otherembodiments of the binding molecule-multienzyme complexes of theinvention for the treatment/prevention of HIV-1 infections, soluble CD4(e.g., TT4) may be used as a binding effector molecule to provide viraltarget specificity. Soluble CD4 (sCD4) fragment of CD4 binds to theHIV-1 virus in a manner similar to the binding of whole CD4 (R. Fisher,et. al. Nature 331 (1988) 76-78). The production of sCD4 is describedamong other places in, P. Maddon, et. al., U.S. Pat. No. 5,126,433. Inorder to provide for the covalent attachement of sCD4 to the remainderof the binding molecule-multienzyme complex, one ore more cysteineresidue may be added to the sCD4 (or CD4) by mutation. Preferably, theadditional cysteine residues are located on side of the CD4 fragmentthat is distal to the HIV-1 binding site; preferred sites forintroducing cysteine residue substitution mutations are at sCD4 aminoacid residue positions 64, 364 or 374 or 377. sCD4 containing additionalcysteine residues may be produced either by in vitro peptide synthesistechniques or by conventional recombinant DNA expression techniques.

Binding molecule-multienzyme complexes, particularly embodimentsemploying a liposome as a joining component, may be adapted for thetreatment of atherosclerosis. LDL has a cholesterol and cholesterolester core surrounded by a phospholipid layer. Into the spherical lipiddroplet four apo B molecules are partially buried and are tetrahedrallylocated. The overall structure is well defined and proteins are orientedso that the lipophilic domain is inside the lipid and hydrophilic partis in the water. LDL is very sensitive and forms by vortexing in vitrosimilar deposits as observed in atherosclerotic plaque (J. C. Khoo, E.Miller, P. McLoughlin and D. Steinberg. 1988. Enchanced MacrophageUptake of Low Density Lipoprotein after Self-Aggregation.Arteriosclerosis 8: 348-358). The lipid part and apolipoprotein B areseparated and most of the protein is denatured and deposited. Lipidsfrom several LDL particles aggregate and form droplets having diametersof 30-400 nm. An antibody which is specific for the denatured LDL can beproduced by a person of ordinary skill in the art. The heterogeneity ofthe plaque can be largely circumvented by the use of liposomal bindingmolecule-multienzyme complexes. Furthermore, because liposome bindingmolecule-multienzyme complexes binding is mediated by multipleantibodies, it is much stronger and more selective than the binding by asingle antibody. Up to 50-60 antibodies can bind the liposome to theplaque without preventing enzymatic reaction. When lipid carbon-carbondouble bonds in LDL are oxidized to aldehydes they form Schiff's baseswith the lysines of apolipoprotein B (W. Palinski in "Cell Interactionsin Atherosclerosis" (Eds. N. J. Severs and H. Robenek), CRC Press, BocaRaton, 1992, p. 1.). These structures in oxidized LDL are antigenic andmay used as targets for antibodies to be used as effector molecules,both binding effector molecules and therapeutic effector molecules.Because this reaction proceeds often many years in the plaque, theconcentration of the antigens is high in contrary to circulating LDLparticles, which are metabolized fast. Significant part of the oxidationis performed by the metalloproteins secreted by macrophages.Unfortunately macrophages are located mainly close to the edge of theplaque and the plaque is not evenly oxidized. Accordingly antibody tooxidized LDL does not bind evenly to the plaque. The enzymes coupled tothe antibody would digest mainly the edges of the plaque therebyamplifying the effect of macrophages. The end result might be therupture of the plaque.

Another possibility is to use an antibody that is even moderatelyspecific for plaque to place oxidizing enzymes near deposited LDL.Circulating LDL might also be oxidized to some degree, but it ismetabolized fast and completely removed in a relatively short period oftime. Antibody-multienzyme complex containing antibody for oxidized LDLand digestive enzymes is then given to the patient. Oxidized LDL is nowevenly distributed and the plaque is also digested evenly. Thus, onetreatment cycle consists of series administration of both the oxidativeand the digestive antibody-enzyme complexes. A chimeric human/murineantibody Z2D3 specific for human atheroma has been prepared (J. Narula,C. Ditlow, F. Chen, H. W. Strauss and B.-A. Khaw. 1992. Non-invasivelocalization of experimental atherosclerotic lesions with murine/humanchimeric antibody Z2D3 F(ab')₂. American Heart Association 66thScientific Meeting, Nov. 16-19, 1992) an may be used as effectormolecule in the binding molecule-multienzyme complexes of the invention.Fibrin is a major noncellular constituent of atherosclerotic plaque.Fibrin is insoluble even, if the surrounding lipids are removed. Fibrinnetwork will also hold the cells immobilized. Accordingly the digestionof fibrin is mandatory. Plasmin is natural fibrin hydrolase and ispreferred also in this connection. The problem with several activeproteases is that they are inhibited in the blood and removed from thecirculation. Obvious method to avoid fast removal is to use plasminogen,which can be activated after the liposome has attached onto the plaque.Activation can be performed using, for instance, urokinase orrecombinant tissue plasminogen activator. These two proteins are alreadyclinically approved for the treatment of acute myocardial infarction.They can be used as such or targeted using separate liposomes. Usingliposomes the enzymatic activity is released only if two different typesof liposomes are simultaneously present. The selectivity is increasedenormously, because only a relatively large surface containing enoughantigens can bind these two liposomes simultaneously. For instance,circulating lipoprotein particles will not be affected. Side effects arereduced, because freely floating liposome does not have proteolyticactivity. The principle of using inactive proenzyme can be extended tophospholipase subscript, which has also an inactive proenzyme. The useof proenzymes is not only advantageous for the clinical use, but alsothe production of proenzymes is much easier than the production ofactual enzymes. The presence of active phospholipase A₂ would bevirtually impossible in the goat milk, while proenzyme is harmless.Cholesterol esters and triglycerides are most insoluble lipidcomponents. By hydrolyzing them by cholesterol ester hydrolase andlipase will produce relatively easily soluble fatty acids andmonoglycerides. Cholesterol is the largest lipophilic component.Moreover it is the most difficult to degrade. Cholesterol oxidase willtransform cholesterol to 4-cholester-3-one, which is relatively polar.Thus oxidized cholesterol should be removed from the plaque.

The principles presented in this application enable the purposefulconstruction of huge molecular assemblies having an exactly knownchemical structure. For example, in Example 6, as shown in FIGS. 5A-D,describes the construction of a supramolecule for capturing virusparticles which would have a molecular weight of about 4,000,000Daltons.

Another aspect of the invention is to provide bindingmolecule-multienzyme complexes adapted so as to mediate the transfer ofpolynucleotides of interest into a host cell, i.e., transfection ortransformation. Binding molecule-multienzyme complexes of the inventionfor cell transfection comprise effect of molecules capable of initiationthe natural internalization machinery of a eukaryotic cell. Sucheffector molecules e.g., antibodies, are capable of binding to cellsurface molecules, e.g., receptors, and preferably cross-linking thereceptors when the effector molecules are components of a bindingmolecule-multienzyme complexes of the invention. A bindingmolecule-multi-enzyme complexes comprising multiple antibodies mayincrease chances of internalization by increasing the concentration ofcross-linked cell-surface molecules. Additionally, sets ofsupramolecular components of the invention may be used to transformcells by employing the internalization machinery of the cell. Forexample, a first supramolecular component consisting of a cell surfacereceptor specific antibody joined to a nucleic acid moiety and a secondsupramolecular component consisting of a second cell surfacereceptor-specific antibody joined to a complementary nucleic acidmoiety. By permitting the first and second supramolecular componentnucleic acids to hybridize to one another after the antibody moietieshave bound to a cell surface, receptor cross-linking, and henceinternalization, may be achieved. Supramolecular assemblies of theinvention may also comprise additional nucleic acids for internalizationinto a host cell of interest. Nucleic acid components of supramolecularassemblies for cell transformation may be detached from thesupramolecular assembly in a variety of ways. As shown in FIG. 15A, thenucleic acid may be detached through the use of restriction enzymes orother nucleases. Additionally, nucleic acid components may detach frombinding molecule-multienzyme complexes through the process of nucleicacid denaturalization, provided the nucleic acids are not covalentlyattached to the assembly. In another embodiment of the subject bindingmolecule-multienzyme complexes for transformation, effector moleculeshaving phospholipase A₂ activity may be used to introduce pores into acell membrane. In other embodiments of the invention, the supramolecularassembly may comprise polyamines, e.g., spermine so as to mediatetransformation.

The large scale solid phase synthesis (e.g., over 1 mmole) ofoligonucleotides is difficult to achieve using previously describedsynthesis methods. A significant problem with large scale synthesis isthe efficient mixing of the heterogeneous system. Silica, polystyrene orother similar solid support particles (typically spherical) modifiedwith polyethyleneoxide chains are commonly used as a support foroligonucleotide synthesis. The growing oligonucleotide chains may formcoils and stacking relationships, even between oligonulceotides onseparate support particles, thereby creating a network that can preventthe efficient entry of reagents. The higher density of these sphericalparticles also makes efficient reaction mixing even more difficult.

Large scale synthesis of oligonucleotide, e.g., 0.1-1 mole, is usefulfor the commercial scale production of supramolecules and supramolecularcomponents of the invention. The following improvements of the currentoligonucleotide synthesis procedure solve the above-described problemssurrounding large scale synthesis of oligonucleotides. First,acetonitrile is replaced with a solvent or solvent mixture that has aspecific density of about one and that is also better able to solvatethe heterocyclic bases of nucleotides than acetonitrile. Suitablesolvents having these desired properties include benzonitrile or amixture of acetonitrile and dichlorobenzene. The density of thesesolvents is compatible with the use of polystyrene or comparable solidsupports. Solid supports will float in these preferred solvents, therebypermitting mixing steps to be easily performed. Another improvement overconventional oligonucleotide synthesis that may be used to effect largescale synthesis is the exposure of the reaction mixture to microwavesduring the coupling step. Microwaves increase molecular rotation andreduce unwanted polynucleotide network formation without subjecting thereaction mixture to excessive heat. An additional improvement overconventional oligonucleotide methods synthesis is instead of monomericamidites, dimeric or trimeric amidites may be used as building blocks.Even larger amidite multimers may be used to construct oligonucleotides;however, monomeric, dimeric and trimeric amidites and their combinationsare preferred. Using dimers and trimers as building blocks requirespreparation of 16 dimer amidites and up to 64 trimer amidites separate.The use of multimeric amidites the number of couplings during automatedsynthesis is decreased significantly and accordingly the yield andpurity is increased. The three above-described oligonucleotideimprovements may be employed separately or in combination with oneanother. A person of ordinary skill in the art will appreciate that anideal combination of the above-described improvements will depend uponthe length of the oligonucleotide described and the scale of thesynthesis.

Another aspect of the invention is to provide methods and compositionsfor the in vivo genetic manipulation of cells in humans and otheranimals. Such genetic manipulation methods and compositions may be usedto modify targeted cells so as to either express or not express a geneof interest. By either expressing or not expressing a selected gene,desirable properties may be added to the targeted cells. The geneticmanipulation methods of the invention comprise the step of administeringan effective amount of a binding molecule-multienzyme complex of theinvention adapted for targeted genetic manipulation. The bindingmolecule-multienzyme complex adapted for targeted genetic manipulationmay use a variety of different molecules as joining components,including liposomes, proteins, nucleic acids and the like. Preferably,the joining component is a liposome. The binding molecule-multienzymecomplex adapted for genetic manipulation comprises one or more effectormolecules for targeting the binding molecule-multienzyme complex tospecific cells of interest. Such effector molecules for targetinginclude, but are not limited to, antibodies for cell surface markersspecific to the cell of interest, ligands for receptors, on the cellsurface, and the like. By using one or more targeting effectormolecules, the binding molecule-multienzymes may be targeted to cells ofinterest. The binding molecule-multienzyme complexes adapted for geneticmanipulation further comprise a genetic manipulation complex. Inembodiments of the invention in which the binding molecule-multienzymecomplex adapted for genetic manipulation employs a liposome as a joiningmolecule, the liposome is filled with a plurality of geneticmanipulation complexes. Genetic manipulation complexes comprise one ormore polynucleotide molecules of a sequence designed for homologousrecombination into a specific chromosomal location or locations. Thegenetic manipulation complex further comprises a DNA motor protein. DNAmotor proteins are described in, among other places Vallee and Sheetz,Science 271:1539-1544 (1996) and Gorlich and Mattaj, Science 1513-1518(1996). DNA motor protein bind to intracellular scaffolding proteinfilaments such as actin or tubulin. DNA motor protein bind to thefilaments and be transported along the filament to the nucleus. Byfollowing the intracellular filament proteins to the nucleus, the motorprotein serves to transport the entire genetic manipulation complex tothe nucleus. The genetic manipulation complex may further comprise aprotein (either antibody or other ligand) for binding to one or morenuclear receptors on the surface of the nuclear membrane, therebyexpediting the translocation of the genetic manipulative complex acrossthe nuclear membrane. The genetic manipulation complex also comprises atleast one DNA binding protein, wherein the DNA binding protein canspecifically bind to a sequence of the polynucleotide of the geneticmanipulation complex. The DNA binding protein may also bind directly toa site or sites on the chromosome of the targeted cells, thus providingfor the specific targeting of the polynucleotide to the site ofinterest. The DNA binding protein serves to bring the polynucleotide ofinterest into close proximity with the targeted chromosomal site,thereby facilitating homologous recombination at the site of interest.The polynucleotide of the genetic manipulation complex may include anyof a variety of sequences useful for genetic manipulation. Suchsequences include, but are not limited to, protein encoding sequences,anti-serve encoding sequences, and disrupted protein encoding sequences.The polynucleotide may further comprise a polyadenylating tail (orsignal) so as to reduce the possibility of destruction by endogenousnucleases.

Binding molecule-multienzyme complexes adapted for targeted geneticmanipulation that employ liposomes as joining components employrelatively small liposomes so as to promote cellular uptake. Generally,liposomes of interest are about 10-80 nm in diameter. Optionally, thesurface of the liposomes may comprise a trypsinogen and/or aprophospholipase (phospholipase A₂ being particularly preferred) , or asimilar proenzyme. The proenzyme may, upon activation, serve tofacilitate the release of the genetic manipulation complex from theliposome.

The invention having been described above may be better understood byreference to the following examples. The following examples are offeredin order to illustrate the invention and should not be interpreted aslimiting the invention

EXAMPLES

1. Illustration of Complementary Nucleic Acid Sequences

Table 1 provides examples of nucleic acid sequences and theircomplementary sequences that may be used in the present invention; theconstruction of complementary nucleic acids is known to the person ofordinary skill in the art.

For the purpose of these examples, complementary chains of nucleic acidsare depicted as an integer and that integer underlined. For example,-(A_(n) -C_(p))_(i) is identified as 1 in Table 1. Its complement,-(T_(n) -G_(p))_(i) is labelled 1. With regard to the indices n, p, qand r used in Table 1, it should be understood that these indices areindependent for each set of complementary nucleic acid chains.

                  TABLE 1                                                         ______________________________________                                                           Complementary Comple-                                      Chain                                                                              Unit          Unit          mentary                                                                              Chain                                 ______________________________________                                        1    --(A.sub.n --C.sub.p)i                                                                      (T.sub.n --G.sub.p)i-                                                                              1                                     2    --(A.sub.n --T.sub.p)i                                                                      (T.sub.n --A.sub.p)i-                                                                       n ≠ p.sup.a                                                                    2                                     3    --(C.sub.n --G.sub.p)i                                                                      (G.sub.n --C.sub.p)i-                                                                       n ≠ p.sup.a                                                                    3                                     4    --(A.sub.n --C.sub.p --G.sub.q)j                                                            (T.sub.n --G.sub.p --C.sub.q)j-                                                                    4                                     5    --(A.sub.n --G.sub.p --C.sub.q)j                                                            (T.sub.n --C.sub.p --G.sub.q)j-                                                                    5                                     6    --(A.sub.n --C.sub.p --T.sub.q)j                                                            (T.sub.n --G.sub.p --A.sub.q)j-                                                                    6                                     7    --(A.sub.n --T.sub.p --C.sub.q)j                                                            (T.sub.n --A.sub.p --G.sub.q)j-                                                                    7                                     8    --(A.sub.n --C.sub.p --A.sub.q --G.sub.r)j                                                  (T.sub.n --G.sub.p --T.sub.q --C.sub.r)j-                                                          8                                     9    --(A.sub.n --G.sub.p --A.sub.q --C.sub.r)j                                                  (T.sub.n --C.sub.p --T.sub.q --G.sub.r)j-                                                          9                                     ______________________________________                                         .sup.a If n = p then the oligonucleotide is selfcomplementary and can be      useful when similar units are coupled together.                          

Many of the examples given herein are provided in order to demonstratethe principles of the invention. Preferably, repeating units areavoided.

DNA and RNA triple helices are also well known and may be used to formthe supramolecular assemblies of the invention. Triple helices mayresult from the association between T....A....T and C....G....C. As aresult, nucleic acid changes, such as those listed in Table 2 can beused to form triple helices to bind different components of asupramolecule. One advantage in using triple helix structures isincreased rigidity. This property can be utilized even after thesupramolecule has been assembled. Triple helix forming oligonucleotidesmay be used as the nucleic acid moieties of the supramolecularcomponents of the invention. Double helical structures, which arecapable of binding to a third oligonucleotide, do so and give rigidityand shape to the supramolecule. The use of triple helices insupramolecular assemblies is demonstrated in FIG. 8.

                  TABLE 2                                                         ______________________________________                                                Center coil  Two outer coils                                          ______________________________________                                        11        --A.sub.h      T.sub.h --                                           12        --G.sub.h      C.sub.h --                                           13        --(A.sub.n --G.sub.p)i                                                                       (T.sub.n --C.sub.p)i-                                14        --(A.sub.n --A.sub.p --G.sub.q)j                                                             (T.sub.n --T.sub.p --C.sub.q)j-                      15        --(A.sub.n --G.sub.p --G.sub.q)j                                                             (T.sub.n --C.sub.p --C.sub.q)j-                      ______________________________________                                    

2. Construction of a Square Planar Supramolecule

FIG. 2(A) depicts the construction of a square planar supramolecule fromfour different components. Component A comprises effector molecule M towhich is attached nucleic acid chains 1 and 2. Component B is formed byattaching nucleic acid chains 1 and 3 to effector molecule N. ComponentC is formed by attaching nucleic acid chains 2 and 3 to effectormolecule P. Component D is formed by attaching two nucleic acid chains 3to effector molecule Q. When components A, B and C are mixed, thecomplementary chains 2 and 2 of components A and C bind and thecomplementary chains 1 and 1 of components A and B bind. When componentD is added, the 3 nucleic acid chains bind to the 3 chains of componentsA and C to form the square supramolecule depicted in FIG. 2(A).

FIG. 2 (B) depicts how the square planar supramolecule can be stabilizedby the addition of complementary nucleic acid chains that bind componentA and C to component B to D. Since the distance between diagonallypositioned effector molecules are 1.41 times the distance betweeneffector molecules on the sides of the square supramolecule, thecomplementary nucleic acid chains used to bind the effector moleculesdiagonal to one another must be at least 1.41 times as long as thecomplementary nucleic acid chains binding the adjacent effectormolecules in order to produce a supramolecular assembly with the desiredshape.

3. Construct of a Tetrahedral Supramolecule

FIG. 2(C) depicts the construction of a tetrahedral supramolecule usingfour components. In order to form a tetrahedral supramolecule componentA is attached to components B, C and D by complementary nucleic acidchains. Similarly, components B, C and D are attached to the componentsby complementary nucleic acid chains.

4. Synthesis of Components of Supramolecules

A. Preparation of Nucleic Acid Chains

Several different high yield strategies for oligonucleotide synthesishave been developed, see, for example, M. J. Gait "OligonucleotideSynthesis, a Practical Approach", IRL Press, Oxford, 1984; J. W. Engelsand E. Uhlman, "Gene Synthesis", Angew. Chem. Int. Ed. Engl. (1989)28:716-724. These methods include the phosphate diester, phosphatetriester, phosphite triester and phosphonate methods. Phosphite triesterchemistry, which utilizes highly reactive phosphoramidites as startingmaterials is currently the most favored method of synthesis (R. L.Letsinger, J. L. Finnan, G. A. Heavner and W. B. Lunsford, "PhosphiteCoupling Procedure for Generating Internucleotide Links", J. Chem. Soc.(1975) 97:3278-3279; L. J. McBride and M. H. Caruthers, "AnInvestigation of Several Deoxynucloeside Phosphoramidites Useful forSynthesizing Deoxyoligonucleotides", Tetrahedron Lett. (1983)24:245-248) Oligonucleotides are most commonly prepared with automatedsynthesis (Beaucage, et al., Tetrahedron Lett. (1981) 22:1859-1862; U.S.Pat. No. 4,458,066). All of the known methods are applicable and willprovide molecular building blocks for the supramolecular assemblyprinciple described in this application.

Enzymatic methods for the production of oligonucleotides may also beused to synthesize the polynucleotide moieties of the supramolecularcomponents of the invention. The polynucleotide moieties may also beproduced in vivo and subsequently cleaved into complementary singlestrands by heating, and separated by preparative electrophoresis orchromatography.

Short oligonucleotides may be coupled together chemically orenzymatically to obtain longer oligonucleotides, see, for example (S. A.Narang, et al., Meth. Enzymol. (1979) 68:90; U.S. Pat. No. 4,356,270);N. G. Dolinnaya, N. I. Sokolova, D. T. Ashirbekova and Z. A. Shabarove,"The use of BrCN for assembling modified DNA duplexes and DNA-RNAhybrids; comparison with water soluble carbodiimide", Nucleic Acid Res.(1991) 19:3067-3072).

B. Preparation of Effector Molecules

Effector molecules, which contain aliphatic amino, dialkylamino,trialkylamino, thiol, formyl oxirane, α-halogenocarbonyl, isothicyanatoor hydroxysuccinimidyl ester groups of similar, may be coupled withsuitably derivatized oligonucleotides using bifunctional spacers.Effector molecules which do not contain groups mentioned above may beactivated so that they contain at least one of these groups forcoupling. Groups that can be activated for coupling, include:carbon-carbon double and triple bonds, halogen, carbonyl, carboxyl andhydroxyl.

The amino acid residue sequence of proteins may altered throughwell-known genetic engineering techniques to as to produce non-naturallyoccurring proteins having the desired biological functions of acorresponding naturally occurring protein, but adapted for coupling tonucleic acid moieties. For example, addition of a cysteine residue,either through substitution or inserting, may add a free thiol group forcoupling to a nucleic acid moiety.

C. Attachment of Nucleic Acids to Effector Molecules

Effector molecules may be attached to nucleic acids by numerous methods,including:

1. The molecular moiety is first attached to a solid support and is usedas a linker for oligonucleotide synthesis. When oligonucleotidesynthesis is completed the molecular moiety is detached from the solidsupport so that it remains covalently coupled with the oligonucleotide.An example of this procedure is a FMOC-protected polypeptide which isfirst synthesized on a solid support so that it has a terminal freeserine hydroxyl group. The oligonucleotide synthesis is started fromthis hydroxyl group.

2. Molecular moieties other than nucleotides may be incorporated insidethe oligonucleotide chain during the synthesis so as to providefunctional groups for coupling to nucleic acids. For example, if thesemolecular moieties have at least two hydroxyl groups, one of which isfree and another which is protected by dimethoxytrityl group, thenconventional oligonucleotide synthesis methods can be used to produce anucleic acid that may readily be coupled to an effector molecule.

3. As a last step of the oligonucleotide synthesis a molecular moietyhaving a suitable functional group for coupling may be attached at theend of the oligonucleotide chain. Again, if this molecular moiety has atleast one hydroxyl group, it can be attached as nucleic acid monomer.This approach is already well known in the literature

4. A molecular moiety having a suitable functional group for couplingmay be attached after the oligonucleotide synthesis is completed andpart or all protecting groups have been removed. Especially molecularmoieties attached using methods 1-3 can contain several functionalgroups which are protected by orthogonal protecting groups. This allowsstepwise removal of protective groups and allows regioselectiveattachment of new molecular moieties.

Methods of attaching enzymes to oligonucleotides that are known to theperson of ordinary skill in the art may be used to produce thesupramolecular components and supramolecular structures of theinvention. Descriptions of such techniques can be found in, for example,Jablonski et al. Nucl. Ac. Res. 14:6115-6128 (1986), Ruth DNA 3:123(1984), Balaguer et al. Anal. Biochem. 180: 50-54 (1989), Balaguer etal. Anal. Biochem. 195: 105-110 (1991), Li et al. Nuc. Ac. Res.15:5275-5287 (1987), Ghosh et al. Anal. Biochem. 178:43-51 (1989),Murakami et al. Nuc. Ac. Res. 14:5587-5595 (1989), and Alves et al.Anal. Biochem. 189:40-50 (1988).

In order to covalently couple an oligonucleotide with a effectormolecule, the oligonucleotide must contain a functional group which hasa high enough reactivity to allow specific reaction at predeterminedsite. This functionality can be introduced into an oligonucleotide chainduring normal automated synthesis, if suitable joint molecules are used.Possible functionalities include amino, dimethylamino, thiol, oxiraneand other groups, which are more reactive than functional groups innucleotides. A different approach is to use biotin-avidin chemistry oranother high affinity specific non-covalent interaction. Several meansof introducing these groups have already been published in theliterature. See, for example, Leary, et al., Proc. Natl. Acad. Sci. USA(1983) 80:4045; Richardson and Gumport, Nucl. Acid Res. (1983) 11:6167;Lenz and Kurz, Nucl. Acid Res. (1984) 12:3435; Meinkoth and Wahl, Anal.Biochem. (1984) 138:267; Smith, et al., Nucl. Acid Res. (1985) 13:2399,J. M. Coull, H. L. Weith and R. Bischoff, Tetrahedron Lett. (1987)27:3991-3994; J. Haralambidis, M. Chai and G. W. Tregar, Nucleic Acid.Res. (1987) 15:4857-4876; B. C. F. Chu and L. E. Orgel, Nuc. Acid Res.(1988) 16:3671-3691. In addition to the added functionality of theoligonucleotide strand, a bifunctional spacer molecule is typically usedto couple oligonucleotide and a effector molecule. Many of these spacersare well known in the literature and are commercially available.

1. Attachment of Nucleic Acids to Peptides

Peptides and peptide analogues are very commonly used as effectormolecules. In order to attach oligonucleotides by normal nucleotidechemistry to a peptide, the peptide should have free hydroxyl groups.Primary hydroxyl groups are preferred. These can be implemented into apeptide by using protected ethanolamine on the carboxyl end and glycolicacid on the amino terminal, instead of an amino acid. As shown in FIG.10, serine moieties can be used to give further attachment sites alongthe peptide backbone.

As shown in FIG. 11, the peptide effector molecule can be branched andused as a multivalent effector structure. Several other multivalenteffector structures are possible such as ethylene glycol dimer, trimer,etc. Ethylene glycol derivatives can be connected to polyalcohol to getmultivalent effector structures. In order to fully exploit the presentinvention, conjugation of several nucleic acid chains to a singleeffector molecule must be possible. The process of combining nucleicacids with polymeric support and with the use of spacer molecules iswell known. Similar chemistry can be used in connection with thisinvention to combine nucleic acid chains with effector molecules such asproteins or polypeptides.

One method for conjugating several nucleic acid chains to a singleeffector molecule is described below. The hydroxyl group of2-(2'-aminoethoxy)ethanol (AAE) is first protected byt-butyldimethylsilylchloride (TBS). The product is coupled withFMOC-t-BOC-L-lysine. FMOC-group is removed and two FMOC-glycines areattached similarly. FMOC-L-glutaminic acid -t-butyl ester is the nextcomponent and will later serve as a branching point (see FIG. 13).Peptide chain is extended with two glycines and one lysine. The aminogroup of the last lysine is reacted with propylene oxide whereby asecondary hydroxyl group is formed. This hydroxyl group is protectedwith acid and base stable trichloroethoxycarbonyl group (Troc).

A shorter peptide based chain is synthesized by starting with Trocprotected 2-(2'- aminoethoxy)ethanol and coupling this with one lysineand two glycines using standard peptide chemistry.

Two peptide chains which are prepared as described above are coupledtogether by forming an amide bond between the free carboxylic group ofglutaminic acid and the end amino group of the glycine in the shorterpeptide. The product which has three branches each having one protectedhydroxyl group needs manipulation of the protecting groups before it iscompatible with oligonucleotide synthesis.

Once the properly protected spacer is prepared, the first preparedoligonucleotide is coupled with phototriester synthesis with the freeprimary hydroxyl group (FIG. 14). The shortest oligonucleotide iscoupled in this stage, whereas the longest oligonucleotide is preparedwith automatic synthesizer. The product is not deprotected or detachedfrom the solid support. The synthesis is continued by adding the"trivalent" spacer, which is already coupled with one oligonucleotide.The free secondary hydroxyl group becomes coupled with theoligonucleotide which is still bound with the solid support. Thus thepeptide spacer is coupled with two oligonucleotide chains.Dimethoxytrityl protecting group of the third hydroxyl group is removedby acid. The automated oligonucleotide synthesis is continued and thethird oligonucleotide chain is constructed. The protecting groups arethen removed and the molecule is detached from the solid support.

D. Assembly of Supramolecule from Components

The hybridization is performed preferably in a aqueous medium containingvarious additives. Additives include, but are not limited to buffer,detergent (0.1% to 1%) , salts (e.g., sodium chloride, sodium citratefrom 0.01 to 0.2M), polyvinylpyrrolidine, carrier nucleic acid, carrierproteins, etc. Organic solvents may be used in conjunction to water,such as alcohols, dimethyl sulfoxide, dimethyl formamide, formamide,acetonitrile, etc. In addition to concentration of the derivatizedoligonucleotides, the temperature can be used to control thehybridization. The optimum temperature for hybridization is 20° C. belowthe melting point of the oligonucleotide. This means that the preferredtemperature for hybridizing 30-mers is typically 40-60° C. For shorteroligonucleotides the temperature is lower and for longeroligonucleotides it is higher. Oligonucleotides containing large portionof cytidine and guanine have higher melting point than theoligonucleotides containing a lot of adenine and thymidine. Detailedformulae for calculating the melting temperature of double strandednucleic acids are well known to the person of ordinary skill in the art.Additionally, melting temperature may readily be calculated usingempirical methods.

5. Example of Antibody-multienzyme Supramolecule

Two current main strategies for drug development for HIV are finding ofreverse transcriptase and HIV protease inhibitors. All four approvedAIDS drugs are reverse transcriptase inhibitors. HIV protease inhibitorsare also promising as drugs, but the rapid mutation of the viralprotease has so far been overwhelming obstacle for the development of acommercial drug.

Embodiments of the supramolecules of the invention that comprise anHIV-antibody and several digestive enzymes can destroy the virusparticle itself. Antibodies have earlier been conjugated with enzymesfor drug use (C. Bode, M. S. Runge and E. Haber in "The Year inImmunology 1989-1990". Molecules and Cells of Immunity (J. M. Cruse andR. E. Lewis, Eds.) Vol. 6, Karger Publishing, Basel, 1990). Typicallythese antibody-enzyme complexes are used to produce active drugs fromprodrugs. This embodiment of the invention is particularly advantageousif the drug of interest is highly toxic at therapeutic levels. Forexample, the drug against cancer can be produced on the surface of thecancer cell and cancer cells are subjected to higher concentration ofthis drug than other cells. Several antibodies specific for cancer cellsare known. Enzymes are targeted to degrade the plasma membrane of thecancer cell and include lipases, proteases and glycosidases.

One strategy is to couple lipid and RNA degrading enzymes to an HIVspecific antibody. Although a virus does not have its own metabolism toserve as a drug target, a virus is unable to heal itself, if part of thevirus is destroyed by externally added catabolic enzymes.

In order for these enzymes to have operational freedom, the spacerbetween the antibody and the enzyme must be of sufficient, e.g, e.g., onthe order of 10 nm. In this case virtually the whole surface of thevirus is covered. In order to avoid allergic reactions this spacer mustbe fully biocompatible, preferably a normal biological component. Inaddition it should have some rigidity to allow structures in whichenzymes and antibodies do not interfere with each other. Because theseantibody-enzyme complexes can be complicated structures, a self-assemblywould be ideal. Oligonucleotides fulfill all these requirements. Furtherrequirement is that joints connecting enzymes and antibody witholigonucleotides are as small as possible to suppress immunoreaction.These drugs are supramolecular drugs, i.e., noncovalent interactions areimportant structural factors. Especially complementary hydrogen bondingof oligonucleotides is essential for the assembly and structuralintegrity.

In FIG. 3A is a schematic representation of one possible supramoleculedemonstrating this principle. Antibody is in central position and fourdifferent enzymes: phospholipase A₂, lipase, cholesterol esterase andribonuclease A. Phospholipase A₂ can be supplemented or completelyreplaced by another phospholipase such as phospholipase C. One extrasingle stranded oligonucleotide is attached with the antibody. Thisoligonucleotide is complementary with viral RNA and binds viral RNA whenvirus is disintegrated.

Many viruses, including HIV-1, are covered by a lipid bilayer which ittakes from the host cell when it is formed. The bilayer containsphospholipids, triglycerides and cholesterol esters. Accordingly threeenzymes specific for these classes of compounds are used to digest theviral lipid bilayer. When the bilayer is hydrolyzed, fatty acids andlysolipids are formed. These digestion products are soluble in bloodplasma and may be bound by albumin, which is a scavenger protein toremove free fatty acids and lysolipids. When the protein core of thevirus is exposed to plasma it is to be expected that the proteindissolves spontaneously and RNA is released. This process happens whenthe virus is internalized into a cell. The lipid bilayer fuses with theplasma membrane of the cell and virus becomes unstable and dissolvesinto the cytoplasm of the cell. No specific endocytosis mechanism hasbeen observed for HIV. In essence our idea is to induce the dissolutionof the virus outside the cell and destroy viral RNA when it is released.In order to promote the breakdown of RNA a short complementaryoligonucleotide is attached with the antibody and also ribonuclease A ispart of the enzyme palette. Proteinases are not included among theenzymes in our first design, because it is feasible to suppose that theprotein effector of the HIV is unstable when exposed. If opposite turnsout to be true, it is possible to include some proteinases. However,blood contains inhibitors against many proteinases, especially ifproteinases are nonspecific. Some specific endopeptidases as well ascarboxypeptidases and aminopeptidases can be used, because they are notinhibited.

A similar strategy can be used for cancer therapy and to remove `plaque`from blood vessels, e.g., to treat atherosclerosis. In each caseantibody must be replaced with another antibody or other recognitionmolecule, which is specific for the target. Also enzymatic compositionmust be adjusted for each application.

The antibody-multienzyme supramolecule is assembled fromoligonucleotide-enzyme conjugates and branched oligonucleotidesaccording to FIG. 3(B). FIG. 3(C) depicts two simplified supramolecules,which together can carry the same enzymes as the supramolecule in FIG.3(A).

An important consideration in the synthesis is the incorporation ofamino or thiol functionalities into a desired point of theoligonucleotide during automated synthesis. Phosphoramidite synthesis isdescribed in 5.1-5.7. Their use in oligonucleotide synthesis isstraightforward. By using amino and thiol specific cross-linking agents,the synthesis of branched oligonucleotides is also easily accomplished.The oligonucleotide strands are by A and B and their complementaryoligonucleotides are denoted by corresponding underlined letters.Enzymes are attached into either 3' or 5'-terminus of theoligonucleotide, which contains an amino group. This kind of coupling ofoligonucleotides and proteins is a standard practice in biochemicalconjugation. Antibody is attached into the center of a oligonucleotidechain containing an aliphatic amino group in that position.

After molecular building blocks are synthesized, the final step is aself-assembly of a supramolecule. This relays on the pairwisecomplementarily of the oligonucleotide strands in the components, whichare designed to bind together. In principle, the components contain thecomplete information of the structure of the final supramolecule and asimple mixing of the component molecules will produce the wantedproduct. However, in order to make certain that the assembly proceeds asdesigned, the stepwise process is to be preferred.

Preparation of the antibody-multienzyme supramolecule

Abbreviations: Aminopropanol, AP; 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite, CEDIPCPA; Dichloromethane, DCM; Di-isopropyl ethylamine, DIPEA; Fluorenylmethoxycarbonyl, FMOC;Fluorenylmethoxycarbonylchloride, FMOCCl; Methanol, MeOH;Monomethoxytrityl, MMT; Monomethoxytritylchloride, MMTCl; Serinol, SER;Tetrahydrofurane, THF; Triethylamine, TEA.

5.1. N-Monomethoxytrityl Aminopropanol (MMT-AP) MMTCl (1.54 g) in 10 mlof DCM was added to a solution of AP (1.5 ml) in 5 ml of DCM. Thereaction mixture was 24 h at +4° C. 10 ml of DCM was added and themixture was washed twice with 10 ml of 5% NaHCO₃ and with 5 ml of water.The DCM phase was dried with solid NaHCO₃. The solution was concentratedinto 5 ml in vacuo and applied to 40 g silica column, which was elutedwith 300 ml of DCM/TEA 200:1, 300 ml of DCM/EtOAc/TEA 200:2:1 and 200 mlof DCM/EtOAc/TEA 100:2:1. 1.32 g of pure MMT-AP was obtained.

5.2. N-Monomethoxytrityl aminopropyl cyanoethylN,N-diisopropylphosphoramidite (MMT-AP-CEDIPPA)

To a solution of MMT-AP (0.42 g) in 8 ml of DCM was added 475 μl ofEDIPA and 0.30 g Of CEDIPCPA in 2.5 ml of DCM. After min the reactionmixture was applied directly to g silica column. The column was elutedwith DCM/EtOAc/EDIPA 98:1:1. Fractions of 6 ml were collected. Theproduct was in fractions 3 and 4. The yield was 0.44 g.

This product was used in oligonucleotide synthesis.

5.3. N-Fluorenylmethoxycarbonyl aminopropanol (FMOC-AP)

FMOCCl (1.55 g) in 10 ml of THF was added into a solution of 0.90 g ofAP in 40 ml of water. After 30 min stirring the reaction mixture wasextracted with 20 ml of DCM. The DCM solution was washed twice with 10ml of water and dried with MgSO₄. The solvent was removed with a rotaryevaporator and the residue was dissolved into 14 ml of EtOH and 14 ml ofwater was added. The small precipitate was filtered off and the solutionwas put into a refrigerator. After 20 h the precipitate was separated byfiltration. The yield was 1.22 g.

5.4. N-Fluorenylmethoxycarbonyl aminopropyl cyanoethyl N,N-diisopropylphosphor-amidite (FMOC-AP-CEDIPPA)

FMOC derivative was done exactly as MMT analog in Example 2 using 0.30 gFMOC-AP. Also purification was done similarly. The product was infractions 3-8. Fractions 3-7 contained 0.41 g product.

This product was used in oligonucleotide synthesis.

5.5. N-Fluorenylmethoxycarbonyl serinol (FMOC-SER)

FMOCCl (1.55 g) in 10 ml of THF was added into a solution of 0.54 g ofSER in 30 ml of water and 8 ml of 1.5-M Na₂ CO₃. After 30 min stirringthe reaction mixture was extracted with 20 ml of EtOAc. The EtOAcsolution was washed twice with 10 ml of water and dried with MgSO₄. Thesolvent was removed with a rotary evaporator and the residue wasdissolved into a mixture of 5 ml of EtOH and 30 ml of DCM. The productcrystallized in +4° C. Yield was 1.12 g.

5.6. N-Fluorenylmethoxycarbonyl O-dimethoxytriphenyl serinol(FMOC-DMT-SER)

FMOC-SER (1.12 g) was dissolved into 6 ml of pyridine and 0.68 g ofsolid DMTrCl was added. The reaction mixture was put into +4° C. After20 h 20 ml of water was added and the oily layer was washed with 5 ml ofwater and dissolved into 10 ml of EtOAc and the solvent was removed invacuo. The residue (1.76 g) was fractionated in 28 g silica column,which was eluted with DCM/EtOAc/MeOH/TIPEA 98:1;0.2:0.5 and 96:4:1:0.5.Yield of pure product was 0.72 g.

5.7. N-FluorenylmethoxVcarbonyl O-dimethoxytriphenyl serinyl cyanoethylN,N-diisopropylphosphoramidite (FMOC-DMT-SER-CEDIPPA)

FMOC-DMT-SER derivative was produced essentially as described for thephosphoramidite in Example 5.2 using 0.65 g FMOC-AP. The product waspurified similarly. The desired reaction product was found in fractions4-9. Fractions 5-8 contained 0.82 g product. TFMOC-DMT-SER may also besynthesized by first protecting serinol with DMT and then with FMOC.This variation allows also acylation of the amino group of serinol withcarboxylic acid carrying various other functionalities, such asprotected amino or thiol groups and biotin.

The desired product was used in automated synthesis to introducealiphatic amino group in the position of 20 in a 51-mer.

5.8. Automated Synthesis of Oligonucleotides

The following oligonucleotides were synthesized by automated synthesis:

A 3'TGGAGATGGGGCACCATGCTX5' (SEQ ID NO:1)

B 3'AGCATGGTGCCCCATCTCCAYAGTCACAGCACAGCACTAATAACAAGAAA5' (SEQ ID NO:2)

C 3'TYTTTCTTGTTATTAGTGCTGTGCTGTGACT5' (SEQ ID NO:3)

D 3'GTGATAGGAGTTGATTACAGTCCTX5' (SEQ ID NO:4)

E 3'AGGACTGTAATCAACTCCTATCACYATCAGAAGAGTGAGACGGTGGGAT5' (SEQ ID NO:5)

F 3'TYATCCCACCGTCTCACTCTTCTGAT5' (SEQ ID NO:6)

G 3'TAGACTCAGCGCAATCGTGAAGCTX5' (SEQ ID NO:7)

H 3'AGCTTCACGATTGCGCTGAGTCTAYGATTCTCGGCTCGTTCGAAGTGTC5' (SEQ ID NO:8)

I 3'TYGACACTTCGAACGAGCCGAGAATC5' (SEQ ID NO:9)

X represents MMT-AP-CEDIPPA (5.2) and Y represents FMOC-DMT-SER-CEDIPPA(5.7). Analogous amidites may also be to introduce aliphatic aminogroups.

5.9. Purification of Monoclonal Antibody

Anti gp41/160 (antibody IAM3D6) supernatant had a concentration of 315mg/l. It was purified in 160 ml portions in Protein A Sepharose FastFlow 5 ml column. The supernatant was buffered with 40 ml of 0.2-M Na₂HPO₄. After feeding the supernatant into the column, the column waswashed with 120 ml of 0.1-M Na₂ HPO₄. The antibody was eluted off thecolumn with 0.1-M citric acid and neutralized immediately with 3-M KOH.The antibody solutions were stored at -18° C.

5.10. Acetylated Protein A Sepharose Gel

Protein A Sepharose was packed into 1.5 ml column. It was saturated byeluting with a solution containing 50 mg of monoclonal antibody (Antigp41/160 IAM3D6) . The column was washed with 0.1-M Na₂ HPO₄ buffer (15ml) and eluted 10 ml 1 mM acetyl N-hydroxy succinimide solution inDMF/water 1:9. The antibody was removed by 0.1-M citric acid. Theacetylated Protein A Sepharose was used to couple antibody withnucleotides and in the final assembly of the supramolecule.

5.11. Coupling of Oligonucleotide with Antibody

A solution of antibody (40 mg/25 ml water) was eluted through the columncontaining 1.5 ml acetylated Protein A Sepharose. The Sepharose waswashed with 3 ml of 0.1-M Na₂ HPO₄ and taken out of the column toperform a bath reaction with derivatized nucleotide.

Oligonucleotide 2 (10 mg, 0.5 μmole), comprising two equal 30-mers boundtogether by an amino group containing joint, was dissolved into 1 ml of0.1-M NaHCO₃ and 50 μl of 1-M solution of bis(hydroxysuccinimidyl)glutarate in acetonitrile was added. After one hour the water solutionwas extracted twice with 1 ml of EtOAc and the solution was dialyzed 2 hagainst 0.1-M NaHCO₃. The activated nucleotide was added into a slurryof Sepharose. The mixture was stirred six hours and packed into acolumn. The antibody coupled to the nucleotide was eluted off the columnwith 0.1-M citric acid. Antibody-oligonucleotide conjugate wasfractionated in a Sephadex G-25 column and antibody connected witholigonucleotide was collected.

5.12. Coupling of Oligonucleotide with Enzymes

Oligonucleotide 1 (20 mg,2 μmole), which was contained aliphatic aminogroup at 5'-position was dissolved into 2 ml of 0.1-M NaHCO₃ and 400 μlof 1-M solution of bis(hydroxysuccinimidyl) glutarate in acetonitrilewas added. After one hour the water solution was extracted twice with 1ml of EtOAc. The solution was dialyzed 2 h against 0.1-M NaHCO₃ and 0.5ml aliquots of this solution were added into the following enzymesolutions:

a. 10 mg phospholipase A₂ in 1 ml of water.

b. 40 mg lipase in 4 ml of water.

c. 10 mg ribonuclease in 1 ml of water

d. 30 mg carboxypeptidase in 3 ml of water

5.13. Assembly of the supramolecule

Antibody connected with oligonucleotide was eluted through a acetylatedProtein A Sepharose column (1.5 ml) so that the column was saturatedwith antibody. The column was thermostable at +40° C. andphospholipase-oligonucleotide conjugate solution (twice the equivalentamount) was circulated through the column and UV-flow cuvette. WhenUV-absorption at 280 nm was decreased into half the ribonucleaseA-oligonucleotide conjugate was circulated similarly through the column.Generally about two hours was needed for a complete reaction. Thesupramolecule was eluted off the column by 0.1-M citric acid andneutralized immediately with 1-M KOH. The other supramolecule depictedin FIG. 3(C) was prepared similarly.

6. Design of Supramolecule for Capturing Virus Particles

This example describes the design of a supramolecular assembly that iscapable of surrounding a comparatively large particle, e.g, a virus.First, a structure, which is capable of performing the desired function,is designed and the geometrical features are fixed. Then chemical andphysical features are chosen based on the application. Hydrophilicity,hydrophobicity, acidity, alkalinity, charge transfer, etc., is mappedonto the structure. This designed structure may be visualized as asingle molecule, although in many instances the synthesis of thismolecule would be difficult to achieve at a reasonable yield. In suchembodiments, supramolecular retrosynthesis is performed, i.e., thestructure is broken down into small molecules, which are capable viaself-assembly of forming the original structure. The supramolecularassembly produced in this manner is not identical with the moleculerepresented as a schematic in the figures; however, the importantcharacteristics, i.e., geometry and chemical and physical propertieslisted above, remain the same. Supramolecular retrosynthesis does nottry to retain the original molecular structure intact, but tries toretain all the important chemical and physical properties of the desiredstructure.

Another retrosynthetic cycle can be performed for the molecules obtainedin the previous retrosynthesis to obtain smaller molecular buildingblocks. Finally, molecules are obtained that can be designed andprepared easily. In the design example given below, there are tworetrosynthetic cycles.

Many viruses have an icosahedral shape. Such a virus can be covered byan icosahedral and assembly designed according to this invention. Thisprocess is demonstrated stepwise in FIGS. 5A-E. Dimensions referencedare taken from HIV (human immunodeficiency virus), but the sameprinciples apply to any virus. In this example, polypeptides andoligonucleotides are used, because synthetic methods are available fortheir high yield synthesis. As synthetic methods further develop,analogues or completely artificial supermolecular systems can be madeusing the same design and construction principles offered by theinvention.

Each edge of HIV is about 80 nm. In preliminary design we suppose thatthree amino acid or nucleic acid residues are needed per nanometer. Thecircles in FIG. 4 represent cyclic polypeptides containing enough lysineso that five polylysine chains can be attached. These polylysines aredenoted by zig-zag lines in FIG. 4. Polylysine should contain about 200residues in order to cover whole edge. Onto the other end of eachpolylysine chain is coupled another cyclic peptide that has fournucleotides attached. These nucleotide strands are denoted by a wavyline in FIG. 4. Two of these oligonucleotides are the same, for example,oligonucleotide 1 (n=p=1, i=100). Two others are mutually complementary,but they are bound to the cyclic peptide so that coupling occurs easilybetween neighbors, but not intramolecularly. Thus, they form a pentagonshaped double helix. FIG. 4 shows single stranded oligonucleotide isbound by polylysine. Another molecule is designed using the sameprinciples, but instead of oligonucleotide 1, the single strandedoligonucleotide is now 1. When either of these molecules encounters avirus, which has a negatively charged surface, polylysine isCoulombically associated with the virus. Simultaneously, a negativelycharged oligonucleotide (e.g., 1 or 1) is released from the polylysine.When a complementary capping molecule is associated with the virus, thecomplementary oligonucleotides (1 and 1) combine to close the cage fromwhich the virus can not escape.

The molecules shown in FIGS. 4-5 and described above would be incrediblydifficult to synthesize. However, by designing a supramolecularconstructed from smaller components, synthesis of a virus capturingmolecule is made possible.

FIG. 6 demonstrates how the analogous structure for the large moleculein FIG. 4 can be prepared using smaller molecules. These smallermolecules are shown separately in FIG. 6. Thus, in this approach sixdifferent compounds are needed to get the overall structure, which issame as that of the molecule in FIG. 4. Four of these are relativelysimple, because in each two oligonucleotides are connected to a spacer,which can be polypeptide. Two molecules in the upper part of FIG. 4 arestill relatively complicated because in both cases five nucleotides areconnected to a cyclic spacer, which can be a cyclic peptide. Theseoligonucleotides are denoted by (3,3,3,3,3) and (1,1,8,7,8) . In thesenotations only free single stranded oligonucleotides are listed. Thesestructures can be synthesized by attaching each type of oligonucleotideneeded to a short peptide, for example, pentapeptide Gly-Ala-Ser-Ala-Glywhich is otherwise protected but the hydroxyl group of serine is free.Nucleotide is connected with this hydroxyl group using normal phosphatecoupling. Then, using peptide synthesis methods, these pentapeptidesconnected to a specific oligonucleotides are coupled in a desired order.Closing the cycle makes the molecule more symmetric, but is notessential for the supramolecular assembly or the function of thisassembly in most cases.

There is a further possibility of assembly of cyclic structurescontaining five oligonucleotide chains by using the general principlesof this application. This second step of supramolecular retrosynthesisis demonstrated in FIG. 8. Both of these cyclic structures can beassembled from five smaller molecules. For (3,3,3,3,3) these moleculesare twice (3,1,2) and once (3,2,4), (3,2,4), (3,1,2) and for (1,1,8,7,8)these are (2,1,6), (6,1,5) , (5,8,4), (4,7,3), (3,8,2). In thesenotations it is immediately clear that molecule (2,1,6) combines withthe molecule (6,1,5), because complementary nucleotides are written lastand first, respectively. Looking at the whole sequence of five moleculesindicates that the notation starts with nucleotide 2 and ends with 2.This means that these ends will bind together and form a pentagon. Afterassembly, a supramolecule is obtained which has the same overall shapeas two molecules in upper part of FIG. 7. These supramolecules are stilldenoted by listing only their single stranded oligonucleotides, becausethis is important for further assembly and is sufficient for purposes ofthis application. The symbols are (3,3,3,3,3) and (1,1,8,7,8). Thesesupramolecules also function similarly in further assembly of thestructure, which has the same shape as the molecule in FIG. 4. Thisdemonstrates that almost any structure can ultimately be created frommolecules which has a spacer or a molecular moiety having an active rolein the final assembly connected to two or three oligonucleotides. Thespacer can be a very small molecule or it can be a large molecule. Thespacer can actually be a DNA strand.

Supramolecular assemblies are preferably prepared in an aqueousenvironment, although some embodiments may be assembled in organicsolvents. When effector molecules are lipophilic, the Langmuir-Blodgetttechnique may be utilized. Stepwise assembly is often advantageous. Forexample, the cyclic structures (3,3,3,3,3) and (1,1,8,7,8) in FIG. 8 areassembled separately. These two structures can be stabilized internallyby cross-linking their double helices. This cross-linking can beperformed in a highly selective manner. By cross-linking, both of thesesupramolecular assemblies become covalent molecules. Cross-linking isnot essential, but can be advantageous, because it increases thermalstability. After first assembly and possible cross-linking, the productcan be purified. Purification as well as cross-linking is to berecommended, if the same oligonucleotide is used in several differentplaces.

During the second assembly step (3,4) and (4,7) (see FIG. 6) are addedto (3,3,3,3,3) to give (7,7,7,7,7). In the third assembly step theproduct (7,7,7,7,7) and (1,1,8,7,8) are combined to form 10*(1,8). Thefourth assembly step is the formation of a pentagon by adding (8,9) and(8,9) to give 10*(1). The fifth and final assembly step is adding singlestranded oligonucleotide 1. and the end product is 10*(1). After eachstep, cross-linking or purification or both can be performed dependingon the final requirements regarding quality of the product. Thecomplementary supramolecule 10*(1) is prepared similarly.

If necessary, the cage surrounding the virus can be made more denseusing the principles of this application. The number of molecules neededis then correspondingly larger.

DNA double helix is thermally unstable and cross-linking may be requiredfor stability. One possible approach is shown in FIG. 12. The last aminoacid residue in the spacer is lysine and a complementary DNA strandcontains an alkylating group, which binds preferentially with the aminogroup of lysine, because it is the most nucleophilic of the functionalgroups in this assembly. Thus, perfect chemical control can bemaintained also in the cross-linking process, although this is notalways necessary and more random cross-linking methods can be used.Incorporating photoactivatable groups, like azido adenosine or bromo- oriodo uridine, into oligonucleotide chains allows photochemicalcross-linking, which is site specific. Also use the 3-thioribose inoligonucleotide and cysteine in the peptide spacer allows formation ofdisulphide bridges.

7. Preparation of Components for Liposomes

Many naturally occurring proteins may be used as such without anymodification. Because in some cases these liposomes are used in vivo inhumans, the human proteins should be used to minimize unwanted hostimmune responses. Human proteins can be produced in transgenic plants oranimals. The amino acid sequence of proteins may also be altered throughwell-known genetic engineering techniques to produce mutated proteinshaving the desired biological functions of corresponding naturallyoccurring protein, but adapted to coupling to lipid molecules. Forexample, addition of a cysteine residue, either through substitution orinserting, will add a free thiol group for coupling to a lipid molecule.Moreover the location of this cysteine can be deliberately chosen. Itshould be located so that conjugation does not disturb the activity ofthe protein. Normally this means that the cysteine must be as far aspossible from the active site.

In order to covalently couple a lipid with an effector molecule, thelipid must contain a functional group which has high enough reactivitywith heterobifunctional cross-linker. Phosphatidylethanol-amines (PE)have free aliphatic amino group and can be used directly forconjugation. The conjugation of phosphatidylethanolamines (PE) andserines (PS) with proteins is well known in the literature (Egger et.al. Biochem. Biophys. Acta 1104 (1992) 45-54). Especially, if theprotein contains a thiol group, a chemically well defined conjugate maybe easily prepared. Several spacers are commercially available to coupletwo molecules, which contain an amino and a thiol group. If a proteindoes not contain an a thiol group, an aliphatic amino group, may be usedfor conjugation. Thiol groups are preferred for conjugation sites inproteins as compared with amino groups because of the lower abundance ofthiol groups in most proteins.

Proteins can also be conjugated with lipids after the preparation of theliposomes so as to position the proteins on the outer surface of theliposome.

Phospholipids having unnatural stereochemistry may be prepared asdescribed in the literature, for example see, (J. A. Virtanen et. al.,Chem. Phys. Lipids 27 (1980) 185). D-Mannitol is tritylated to yield1,6-ditrityl-D-mannitol, which is oxidized with lead tetra-acetate andthe product reduced with sodium borohydride to yield1-trityl-sn-glycerol (1-TrG). Diacylglycerols and phospholipids can beprepared starting from 1-TrG by standard methods. For the liposomesdescribed in this application ether bond is preferred at least in theprimary hydroxyl group, because it is resistant against lipase. insn-hydroxyl ester bond can be used, because unnatural stereochemistrywill make this bond stable against phospholipase A2. Instead ofglycerophospholipids sphingolipids or completely artificial lipids,which are resistant against these enzymes, can be used.

8. Examples of effector molecule combinations for specific diseases

Binding molecule-multienzyme complexes may be designed so as to treatdiseases caused by specific pathogenic agents. Examples of effectormolecule combinations are provided below in table 3 so that from each ofthe three columns (titled:antibodies, enzymes and peptides) suitableeffector molecules are chosen. Binding molecule-multienzyme complexes,as described in this example, may contain one or several antibodies,which can be different or the same as one another. The bindingmolecule-multienzyme complexes may comprise 3-8 different enzymes astherapeutic effector molecules, which are typically selected so that 1-3of the enzymes degrade lipids, 0-3 of the enzymes degrade proteins, 1-2of the enzymes degrade nucleic acids and 0-3 of the enzymes degradecarbohydrates. Chemotactic peptides may be used as effector moleculesenhance the natural defence mechanisms of the organism for treatment.Platelet aggregation inhibitors may also be used as effector moleculesto prevent bacterial adhesion to human cells. Additionally, componentssuch as coenzymes, cholic acids, polyamines and metal chelates can beincluded. These additional compounds may be used to activate enzymes orfacilitate accessibility to the binding target so that enzymatictherapeutic effector molecules may more readily degrade the target. Thebinding molecule-multienzyme complexes may be also loaded into anotherassembly so that enzymes are activated at the target site. For example,instead of active enzymes, proenzymes may be used. When proenzymes areemployed as therapeutic effector molecules. In this case a small amountof activating enzyme is brought into the target.

                  TABLE 3                                                         ______________________________________                                        Antibodies:                                                                             Enzymes:       Peptides:                                            ______________________________________                                        VIRUSES                                                                       Anti gp41 (HIV)                                                                         Phospholipase A.sub.2                                                                        Chemotactic                                          Anti gp120 (HIV)                                                                        Phospholipase C                                                                              peptides, e.g.,                                                Lipase         N-Formyl-Met                                         Anti hepatitis B                                                                        Cholesterol esterase                                                                         Leu--Phe                                                       Cholesterol oxidase                                                           Aminopeptidase                                                                Endoproteinase Arg--C                                                         Endoproteinase Asp--N                                                         Endoproteinase Lys--C                                                         Carboxypeptidase A                                                            Carboxypeptidase B                                                            Chymotrypsin                                                                  Ribonuclease A                                                                Ribonuclease B                                                                Ribonuclease C                                                      BACTERIA, PROTOZOA AND FUNGI                                                  Anti TB   α-Amylase                                                                              Chemotactic                                          ( Mycobacterium                                                                         β-Amylase peptides, e.g.,                                      tuberculosis)                                                                           Galactosidase  N-Formyl-Met--                                                 Galactose oxidase                                                                            Leu--Phe                                             Anti Syphilis                                                                           α-Mannosidase                                                 (Treponema                                                                              β-Mannosidase                                                                           Platelet aggre-                                      pallidum)                gation inhibitors,                                             Phospholipase A.sub.2                                                                        e.g., Arg--Gly--Asp--                                Anti Cholera                                                                            Phospholipase C                                                                              Ser                                                  ( Vibrio  Lipase                                                              Cholerae) Cholesterol esterase                                                          Lysozyme                                                                      Lactoferrin                                                         Anti plasmodium                                                                         Aminopeptidase                                                                Endoproteinase Arg--C                                                         Endoproteinase Asp--N                                                         Endoproteinase Lys--C                                                         Carboxypeptidase A                                                            Carboxypeptidase B                                                            Chymotrypsin                                                        CANCER                                                                        HUMAbSK1  α-Amylase                                                                              Chemotactic                                                    β-Amylase peptides, e.g.,                                      Humanized Galactosidase  N-Formyl-Met--                                       antimucin Galactose oxidase                                                                            Leu--Phe                                                       α-Mannosidase                                                           β-Mannosidase                                                                           Platelet aggre-                                                               gation inhibitors,                                             Phospholipase A.sub.2                                                                        e.g., Arg--Gly--Asp--                                          Phospholipase C                                                                              Ser                                                            Lipase                                                                        Cholesterol esterase                                                          Aminopeptidase                                                                Endoproteinase Arg--C                                                         Endoproteinase Asp--N                                                         Endoproteinase Lys--C                                                         Carboxypeptidase A                                                            Carboxypeptidase B                                                            Chymotrypsin                                                                  Alkaline phosphatase                                                          Polyphenol oxidase                                                  ATHEROSCLEROSIS                                                               Murine/Human                                                                            Phospholipase A.sub.2                                                                        Chemotactic                                          IgG1Z2D3  Phospholipase C                                                                              peptides, e.g.,                                                Lipase         N-Formyl-Met--                                                 Cholesterol esterase                                                                         Leu--Phe                                                       Cholesterol oxidase                                                           Aminopeptidase Platelet aggre-                                                Endoproteinase Arg--C                                                                        gation inhibitors,                                             Endoproteinase Asp--N                                                                        e.g., Arg--Gly--Asp--                                          Endoproteinase Lys--C                                                                        Ser                                                            Carboxypeptidase A                                                            Carboxypeptidase B                                                            Chymotrypsin                                                                  Collagenase                                                         ______________________________________                                    

9. Examples of liposome formulation adapted for the treatment of HIVinfections

ABBREVIATIONS

Buffers:

BA: 10 mM NaH₂ PO₄ +0.1M NaCl

BB: 10 mM Na₂ HPO₄ +0.1M NaCl

BD: 0.25M dithiotreitol in BA

MOPS: 3-(N-morpholino) propanesulfonic acid

PBS: Standard phosphate saline buffer

Spacers and conjugation agents:

MIPEG: Maleimide polyethyleneglycol

BisMIPEG: Bismaleimide polyethyleneglycol

SPDP: 3-(2-Pyridyldithio) propionic acid N-hydroxysuccinimide ester

Enzymes:

PLAse: Phospholipase A₂

RNAse: Ribonuclease A

Phospholipids:

OSPC: 1-Octadecyl-2-stearoyl phosphatidylcholine

OSPE: 1-Octadecyl-2-stearoyl phosphatidylethanolamine

OSPE-PDP: N-[3-(2-Pyridyldithio) propionyl]-OSPE

ANTIBODY REDUCTION AND MIPEG-Ab

35 mg (0.25 μmoles) Ab in 5 ml of PBS is dialyzed 3×2 h against 19:1BA/BB at RT.

Into this solution is added 0.5 ml of 0.1M thioethanolamine in 9:1BA/BB.

Kept 1 h at 37° C.

Dialyzed under nitrogen 3×2 h against 9:1 BA/BB, which is purged withnitrogen.

A 50 μl sample diluted with 2 ml of BA and UV-spectrum is measuredbetween 240-400 nm. The absorbance at 280 nm should be about 0.21. 50 μlof SPDP is added. Absorbance is easured at 343 nm immediately after theaddition and after 10 min. The increase in the A₃₄₃ should be about0.024.

160 μl of 50 mM Bis MIPEG in EtOH (8 μmoles) is added and let stand 2 hunder nitrogen at RT.

Dialyzed 3×2 h against 9:1 BA/BB.

The A₂₈₀ should be about 0.20 and A₃₀₅ should be about 0.025.

The MIPEG-PLAse solution is transferred into a bottle.

THIOL-CD4 AND MIPEG CD4

54 mg (1 μmol) of sCD4 in 6 ml is dialyzed 3×2 h against BB in dialysistube (MWCO 15,000).

2.5 μmoles of SPDP in 250 μl of EtOH added and allowed to stand 3 h atRT.

Dialyzed 2×2 h against BA.

A 200 μl sample diluted with 1.8 ml of BA and UV-spectrum is measuredbetween 240-400 nm. The absorbance at 280 nm should be about 0.64. 50 μlof BD is added. Absorbance is measured at 343 nm immediately after theaddition and after 10 min. The increase in the A₃₄₃ should be about0.10.

1 ml of BD is added into the main mixture and let stand 30 min at RT.

Dialyzed 3×2 h against BA. Each dialysis is performed against nitrogenpurged BA and under nitrogen.

A 200 μl sample diluted with 1.8 ml of BA and UV-spectrum is measuredbetween 240-400 nm. The absorbance at 280 nm should be about 0.48. 50 μlof 10 mM SPDP in EtOH is added. Absorbance is measured at 343 nmimmediately after the addition and after 10 min. The increase in theA₃₄₃ should be about 0.06.

Meanwhile, 6 μmoles of BisMIPEG is dried under nitrogen blow.

CD4 solution is added onto BisMIPEG. The solution is purged gently withnitrogen and let stand under vacuum 2 h.

The reaction mixture is transferred into the dialysis tube (MWCO 15,000)and dialyzed against nitrogen purged BA and under nitrogen overnight at+4° C.

25 μl 20 mM MIPA solution is added and dialysis is continued 2×2 hagainst BA under ambient.

The MIPEG-CD4 solution is transferred into a bottle.

MIPEG- PLAse

50 mg (4 μmoles) PLAse is dissolved into 4 ml of 15 mM MOPS and dialyzed2 h against 15 mM MOPS at RT.

Into this solution is added:

40 MG (20 μmoles) of MIPEG

56 mg (160 μmoles) of hexadecylphosphorylcholine (HDPC) and mixed gentlyuntil HDPC is completely dissolved (as micelles).

As a solid are added:

7.2 mg (60 μmoles) of N-hydroxysuccinimide (NHS)

57 mg (300 μmoles) of 1-Ethyl-3-(3-dimethylaminopropyl)carbodimidemethiodide (EDC) and mixed shortly and let stand at RT 6 h or more (upto 16 h).

Dialyzed 3×2h against 9:1 BA/BB.

A 100 μl sample diluted with 1.9 ml of BA and UV-spectrum is measuredbetween 240-400 nm. The absorbance at 280 nm should be about 0.24.

The MIPEG-PLAse solution is transferred into a bottle.

MIPEG-Lipase

50 mg (=1 μmole) lipase is dissolved into 4 ml of BB and dialyzed 2 hagainst BA at RT.

A 200 μl sample diluted with 1.8 ml of BA and UV-spectrum is measuredbetween 240-400 nm. The absorbance at 280 nm should be about 0.27.

Meanwhile 5 μmoles of BisMIPEG is dried under nitrogen blow.

Lipase solution is added onto BisMIPEG. The solution is purged gentlywith nitrogen and let stand under vacuum 2 h.

The reaction mixture is transferred into the dialysis tube (MWCO 15,000)and dialyzed against nitrogen purged BA and under nitrogen overnight at+4° C.

25 μl 20 mM MIPA solution is added and dialysis is continued 2×2 hagainst BA under ambient.

The MIPEG-Lipase solution is transferred into a bottle.

THIOL-RNAse AND MIPEG-RNAse

55 MG (4 μmoles) RNAse is dissolved into 4 ml of BB and dialyzed 2 hagainst BB at RT.

8 μmoles of SPDP in 0.8 ml of EtOH added and allowed to react 2 h at RT.

Dialyzed 2×2 h against BA.

A 200 μl sample diluted with 1.8 ml of BA and UV-spectrum is measuredbetween 240-400 nm. The absorbance at 280 nm should be about 0.16. 50 μlof 10 mM SPDP in EtOH is added. Absorbance is measured at 343 nmimmediately after the addition and after 10 min and 20 min. The increasein the A₃₄₃ should be about 0.09 in 10 min and 0.15 in 20 min.

Meanwhile 18 μmoles of BisMIPEG is dried under nitrogen blow.

CD4 solution is added onto BisMIPEG. The solution is purged gently withnitrogen and let stand under vacuum 2 h.

The reaction mixture is transferred into the dialysis tube (MWCO 15,000)and dialyzed against nitrogen purged BA and under nitrogen overnight at+4° C.

25 μl 20 mM MIPA solution is added and dialysis is continued 2×2 hagainst BA under ambient.

The MIPEG-RNAse solution is transferred into a bottle.

PREPARATION OF LIPOSOMES

The following components are dissolved into 10 ml of 5:5:0.1dichloromethane/ethanol/water:

    ______________________________________                                        47 mg         (66 μmoles)                                                                            OSPC                                                47 mg         (66 μmoles)                                                                            Sphingomyclin                                        8 mg         (12 μmoles)                                                                            OSPE                                                 3 mg         (3 μmoles)                                                                             OSPE-PDP                                            58 mg        (150 μmoles)                                                                            Cholesterol                                         ______________________________________                                    

The mixture is heated at 50° C. in a wide-neck flask under nitrogenblow, while simultaneously adding dropwise dichloromethane to preventcrystallization of any of the components. When almost all solvent hasevaporated, the dichloromethane addition is stopped and nitrogen blow iscontinued 20 min at 50° C. Onto semisolid residue is added 27 ml of warm(50° C.) 9:1 BA/BB.

The mixture is sonified 2 min with Branson tip sonifier using output of4.

A 40 μl sample diluted with 2 ml of BA and UV-spectrum is measuredbetween 240-400nm. The absorbance at 280 nm should be about 0.40.

The Liposome solution is transferred into a bottle.

CONJUGATION OF THE PROTEINS WITH THE LIPOSOME

2 ml of the liposome solution is filtered through 0.2 μm filter (usinghigh pressure filtration will minimize the loss of liposomes). Into thefiltered liposome solution is added 0.2 ml of BD. Let stand 30 min at RTand dialyzed under nitrogen 3×2 h against 9:1 BA/BB.

Into the dialyzed liposome solution is added:

0.35 ml MIPEG-Ab

0.25 ml MIPEG-CD4

0.1 ml MIPEG-PLAse

0.2 ml MIPEG-Lipase

0.1 ml MIPEG₋₋ RNAse

Let stand under nitrogen 4 h at RT.

Fractionated in Bio-Gel A5m column (1 cm×30 cm) eluted with 1 mM 2:3NaH₂ PO₄ /Na₂ HPO₄ +0.1M NaCL. First peak is the liposomal drug elutedin about 8 ml.

The absorbance at 280 nm should be about 0.40.

The Liposomal drug solution is transferred into a bottle.

DOSAGE

About 90% of the mass of the liposomal drug consists of phospholipidsand the rest of the five proteins. In the following, the total mass isindicated.

The minimum and maximum doses are estimated to be 9 mg and 300 mg,respectively. The most probable dose is about 50 mg. If the drug curesthe patient, it is given every day during about two weeks. If it is notcured, it is given every other day or twice a week continuously.

Incorporation by Reference

All patents, patents applications, and publications cited areincorporated herein by reference.

Equivalents

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. Indeed, variousmodifications of the above-described makes for carrying out theinvention which are obvious to those skilled in the field of molecularbiology, organic chemistry, or related fields are intended to be withinthe scope of the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 9                                               - <210> SEQ ID NO 1                                                           <211> LENGTH: 21                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: "Where N = MMt-AP-C - #EDIPPA = N-Monomethoxytrit    yl                                                                                  aminopropyl cyanoethyl N,                                                     N-diisopropylphosphoramidite"                                           <223> OTHER INFORMATION: Chemically Synthesized                               - <400> SEQUENCE: 1                                                           #21                tgct n                                                     - <210> SEQ ID NO 2                                                           <211> LENGTH: 50                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: "Where n = FMOC-DMT - #-SER-CEDIPPA = N-                   Fluorenylmethoxycarbonyl o-dimethoxytriphenyl                                 serinyl cyanoethyl N,                                                         N-diisopropylphosphoramidite"                                           <223> OTHER INFORMATION: Chemically Synthesized                               - <400> SEQUENCE: 2                                                           #              50tctcca nagtcacagc acagcactaa taacaagaaa                      - <210> SEQ ID NO 3                                                           <211> LENGTH: 31                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: "Where n = FMOC-DMT - #-SER-CEDIPPA =                      N-Fluorenylmethoxycarbonyl O-dimethoxytriiphe - #nyl                          srinyl cyanoethyl N,N-diisoopropylphosphor - #amidite"                  <223> OTHER INFORMATION: Chemically Synthesized                               - <400> SEQUENCE: 3                                                           #          31      tgct gtgctgtgac t                                          - <210> SEQ ID NO 4                                                           <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: "Where n = FMOC-DMT - #-SER-CEDIPPA =                      N-Flourenylmethoxycarbonyl O-dimethoxytriphey - #nyl                          serinyl cyanoethy N,N-diisopropylphosphora - #midite"                   <223> OTHER INFORMATION: Chemically Synthesized                               - <400> SEQUENCE: 4                                                           #               25 acag tcctn                                                 - <210> SEQ ID NO 5                                                           <211> LENGTH: 49                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: "Where n = FMOC-DMT - #-SER-CEDIPPA =                      N-Monomethoxytrityl aminopropyl cyanoethyl                                    N,N-diisopropylphosphoramidite"                                         <223> OTHER INFORMATION: Chemically Sythesized                                - <400> SEQUENCE: 5                                                           #               49tccta tcacnatcag aagagtgaga cggtgggat                       - <210> SEQ ID NO 6                                                           <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: "Where n = MMT-AP-C - #EDIPPA = N-Monomethoxytrit    yl                                                                                  aminopropyl cyanoethyl                                                        N,N-diisopropylphosphoramidite"                                         <223> OTHER INFORMATION: Chemically Synthesized                               - <400> SEQUENCE: 6                                                           #              26  ctct tctgat                                                - <210> SEQ ID NO 7                                                           <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: " Where n = MMMT - #-AP-CEDIPPA = N-Monomethoxytr    ityl                                                                                aminopropyl cyanoethyl N,                                                     N-diisopropylphosphoramidite"                                           <223> OTHER INFORMATION: Chemically Synthesized                               - <400> SEQUENCE: 7                                                           #               25 gtga agctn                                                 - <210> SEQ ID NO 8                                                           <211> LENGTH: 49                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: "Where n = FMOC-DMT - #-SER-CEDIPPA =                      N-Fluroenylmethoxycarbonyl O-dimethoxytriphen - #yl                           serinyl cyanoethyl N,                                                         N-diisopropylphosphoramidite"                                           <223> OTHER INFORMATION: Chemically Synthesized                               - <400> SEQUENCE: 8                                                           #               49ctgag tctangattc tcggctcgtt cgaagtgtc                       - <210> SEQ ID NO 9                                                           <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: "Where n = FMOC-DMT - #-SER-CEDIPPA =                      N-Fluorenylmethoxycarbonyl O- dimethoxytri - #phenyl                          serinyl cyanoethyl N,N-diisopropylphophora - #miidite"                  <223> OTHER INFORMATION: Chemically Synthesized                               - <400> SEQUENCE: 9                                                           #              26  gccg agaatc                                                __________________________________________________________________________

What is claimed is:
 1. A targetable antiviral supramolecule comprising:afirst component comprising a target-binding peptide or polypeptide withviral specificity bound to spectrin, and a second component comprising atherapeutic effector polypeptide bound to spectrin, wherein said firstand second components are noncovalently bound through spectrin.
 2. Theantiviral supramolecule of claim 1, wherein said target-binding peptideor polypeptide is glycosylated.
 3. The antiviral supramolecule of claim1, wherein said therapeutic effector polypeptide is an enzyme.
 4. Theantiviral supramolecule of claim 3, wherein said therapeutic enzyme isselected from the group consisting of glycosidases, phospholipases,lipases, cholesterol esterases, nucleases, and proteases.
 5. Theantiviral supramolecule of claim 3, wherein said therapeutic enzyme is aglycosidase.
 6. The antiviral supramolecule of claim 3, wherein saidtherapeutic enzyme is a phospholipase.
 7. The antiviral supramolecule ofclaim 3, wherein said phospholipase is phospholipase A₂.
 8. Theantiviral supramolecule of claim 3, wherein said phospholipase isphospholipase C.
 9. The antiviral supramolecule of claim 3, wherein saidtherapeutic enzyme is a lipase.
 10. The antiviral supramolecule of claim3, wherein said therapeutic enzyme is a cholesterol esterase.
 11. Theantiviral supramolecule of claim 3, wherein said therapeutic enzyme is anuclease.
 12. The antiviral supramolecule of claim 11, wherein saidnuclease is a ribonuclease.
 13. The antiviral supramolecule of claim 12,wherein said ribonuclease is selected from the group consisting ofribonuclease A, ribonuclease B, and ribonuclease C.
 14. The antiviralsupramolecule of claim 12, wherein said ribonuclease is ribonuclease A.15. The antiviral supramolecule of claim 12, wherein said ribonucleaseis ribonuclease B.
 16. The antiviral supramolecule of claim 13, whereinsaid ribonuclease is ribonuclease C.
 17. The antiviral supramolecule ofclaim 3, wherein said therapeutic enzyme is a protease.
 18. Theantiviral supramolecule of claim 3, wherein said target-binding peptideor polypeptide is an Fc receptor.
 19. The antiviral supramolecule ofclaim 3, further comprising a second target-binding peptide orpolypeptide.
 20. The antiviral supramolecule of claim 3, wherein saidtarget-binding peptide or polypeptide is specific for an envelopedvirus.
 21. The antiviral supramolecule of any one of claims 3-17,wherein said target-binding peptide or polypeptide is specific forHIV-1.
 22. The antiviral supramolecule of claim 21, wherein saidtarget-binding peptide or polypeptide is specific for HIV-1 gp120. 23.The antiviral supramolecule of claim 21, wherein said target-bindingpeptide or polypeptide is specific for HIV-1 gp41/160.
 24. The antiviralsupramolecule of claim 21, wherein said target-binding peptide orpolypeptide includes CD4.
 25. The antiviral supramolecule of claim 3wherein said target-binding peptide or polypeptide is specific for aherpesvirus.
 26. The antiviral supramolecule of claim 3 wherein saidtarget-binding peptide or polypeptide is specific for an influenzavirus.
 27. The antiviral supramolecule of claim 3 wherein saidtarget-binding peptide or polypeptide is specific for a hepatitis virus.28. A pharmaceutical composition comprising the targetable antiviralsupramolecule of any one of claims 2-20 and a pharmaceuticallyacceptable carrier.
 29. A pharmaceutical composition comprising theantiviral supramolecule of claim 21 and a pharmaceutically acceptablecarrier.
 30. A method of treating or preventing viral illness comprisingadministering an effective antiviral amount of the pharmaceuticalcomposition of claim
 28. 31. A method of treating or preventing HIV-1infection comprising administering an effective antiviral amount of thepharmaceutical composition of claim 28.